Pain signaling molecules

ABSTRACT

The invention relates generally to novel genes expressed in normal but not Neurogenin-1-deficient animals. The invention relates specifically to a novel family of G protein-coupled receptors and a novel family of two-transmembrane segment proteins that are expressed in dorsal root ganglia, and a method of screening for genes specifically expressed in nociceptive sensory neurons.

This application claims priority under 35 U.S.C. §120 as a continuationof U.S. patent application Ser. No. 10/957,135, filed Sep. 30, 2004 nowabandoned, which in turn is a continuation of U.S. patent applicationSer. No. 10/183,116, filed Jun. 26, 2002 now abandoned, which in turn isa continuation-in-part of U.S. patent application Ser. No. 09/849,869,filed May 4, 2001 now abandoned, which is a continuation of U.S. patentapplication Ser. No. 09/704,707, filed Nov. 3, 2000 now U.S. Pat. No.7,691,604 and under 35 U.S.C. §119(e) to U.S. Provisional Applications60/202,027, filed May 4, 2000, 60/222,344, filed Aug. 1, 2000, and60/285,493, filed Apr. 19, 2001, which are herein incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to novel genes expressed in normal butnot Neurogenin-1-deficient animals. The invention relates specificallyto a novel family of G protein-coupled receptors and a novel family oftwo-transmembrane segment proteins that are expressed in dorsal rootganglia, and a method of screening for genes specifically expressed innociceptive sensory neurons.

2. Description of the Related Art

The treatment of acute and chronic intractable pain is a major target ofdrug development in the pharmaceutical industry. Pain sensation ismediated by primary sensory neurons in the dorsal root ganglia (DRG),which project peripherally to the skin and centrally to the spinal cord.These neurons express signaling molecules, such as receptors, ionchannels and neuropeptides, which are involved in pain sensation. Oneexample is the so-called Vanilloid Receptor-1 (VR-1), which is activatedby capsaicin (chili pepper) as well as by heat and acid. Such painsignaling molecules may also influence pain sensation indirectly byacting as positive or negative modulators of the sensory pathway.Searching for drugs that interact with such signaling molecules, forexample as receptor agonists or antagonists, is an important approach tothe discovery of new therapeutics for the treatment of pain. Newcandidate signaling molecules expressed by pain-sensing (“nociceptive”)sensory neurons are therefore highly desirable targets for new drugscreening and drug discovery efforts. The present inventors havepreviously identified a novel family of basic helix-loop-helix (bHLH)transcription factors, called the Neurogenins (Ngns), which areessential for the development of sensory neurons in the DRG. DifferentNgns are required for the development of different subsets of sensoryneurons. In particular, Ngn1 is necessary for the development of most ifnot all nociceptive sensory neurons. In Ngn1^(−/−) mutant mice, althoughDRG are still present, they are reduced in size and the majority ofnociceptive neurons, identified by expression of markers such as trkAand VR-1, are missing (Ma et al. Genes & Develop, 13: 1717-1728,(1999)). These results suggested that the isolation of genes expressedin wild-type (normal) but not Ngn1^(−/−) DRG might lead to theidentification of novel drug target molecules expressed indifferentiating or mature nociceptive sensory neurons.

While pain is usually a natural consequence of tissue injury, as thehealing process commences the pain and tenderness associated with theinjury resolve. However, some individuals experience pain without anobvious injury or suffer protracted pain after an initial insult. Inaddition, chronic or intractable pain may occur in association withcertain illnesses, such as, for example, bone degenerative diseases,terminal cancer, AIDS, and Reflex sympathetic dystrophy (RSD). Suchpatients may be unable to receive relief with currently-availablepain-relieving (anti-nociceptive) drugs, such as opioid compounds, e.g.morphine, due to problems such as dependence and tolerance. Therefore,there is a great need for novel therapeutic agents for the treatment ofpain, in particular chronic pain.

SUMMARY OF THE INVENTION

The present inventors have carried out a screen for genes expressed inwild-type but not Ngn1^(−/−) DRG using positive selection-baseddifferential hybridization. This screen has identified both knownsignaling molecules involved in nociceptive neuron function, such asVR-1, and novel signaling molecules that are highly specificallyexpressed in nociceptive sensory neurons. The present inventiontherefore includes the discovery of new genes that are expressed innormal mice but not in Ngn1 null mutant mice. One family of novel genesisolated from the screen encodes a receptor protein with 7 transmembranesegments, mrg, a characteristic of G protein-coupled receptors.Subsequent staining experiments (see FIG. 2, 2A-D) confirmed that mrggenes were expressed specifically in subsets of nociceptive neurons inDRG. Another novel gene family isolated in this screen, drg-12, encodesa protein with two transmembrane segments.

In particular, the invention includes isolated nucleic acid moleculesselected from the group consisting of an isolated nucleic acid moleculecomprising a sequence having at least 70% sequence identity to a nucleicacid molecule that encodes the MrgD polypeptide with the amino acidsequence of SEQ ID NO: 49, isolated nucleic acid molecules thathybridize to the complement of a nucleic acid molecule comprising asequence having at least 70% sequence identity to a nucleic acidmolecule that encodes the MrgD polypeptide with the amino acid sequenceof SEQ ID NO: 49, an isolated nucleic acid molecule that that hybridizesunder stringent conditions to a nucleic acid molecule that encodes theMrgD polypeptide of SEQ ID NO:49 and an isolated nucleic acid moleculethat hybridizes to the complement of a nucleic acid molecule thatencodes the MrgD polypeptide of SEQ ID NO: 49.

The present invention also includes the nucleic acid molecules describedabove operably linked to one or more expression control elements,including vectors comprising the isolated nucleic acid molecules. Theinvention further includes host cells transformed to contain the nucleicacid molecules of the invention and methods for producing a proteincomprising the step of culturing a host cell transformed with a nucleicacid molecule of the invention under conditions in which the protein isexpressed. The host cells may be prokaryotic cells, such as E. coli oreukaryotic cells, such as hamster embyonic kidney (HEK) cells or yeastcells.

The invention further provides an isolated Mrg polypeptide selected fromthe group consisting of isolated polypeptides encoded by the isolatednucleic acids described above and the human MrgD polypeptide of SEQ IDNO: 35.

The MrgD polypeptide may be fused to a heterologous amino acid sequence,such as an eptiope tag sequence or an immunoglobulin constant domainsequence.

The invention further provides an isolated antibody that specificallybinds to a polypeptide of the invention, including monoclonal andpolyclonal antibodies, antibody fragments and humanized antibodies.

In another aspect, the invention provides a composition of mattercomprising an MrgD polypeptide or an anti-MrgD antibody in admixturewith a pharmaceutically acceptable carrier. An article of manufacture isalso provided comprising the composition of matter, a container, andinstructions for using the composition of matter to alter sensoryperception in a mammal.

In a further aspect, the invention provides a method of identifying acompound that can be used to alter pain perception in a mammal. Testcompounds are contacted with at least a portion of an MrgD polypeptideof the invention. The MrgD polypeptide or the test compound may beattached to a solid support, such as a microtiter plate. In addition,either the test compound or the MrgD polypeptide is preferably labelled.

Test compounds that are able to form complexes with the MrgD polypeptideare identified. The effects of these compounds is measured in an animalmodel of pain and compounds that alter pain perception in the animalmodel are identified as useful in altering pain perception in a mammal.The compound may enhance or decrease the perception of pain.

In one embodiment the MrgD polypeptide is a native human MrgDpolypeptide, preferably the MrgD polypeptide of SEQ ID NO: 35.

In another embodiment the MrgD polypeptide may be apresent in a cellmembrane or a fraction of a cell membrane prepared from cells expressingthe MrgD polypeptide. In a further embodiment, the MrgD polypeptide ispresent in an immunoadhesin.

The test compounds are preferably selected from the group consisting ofpeptides, peptide mimetics, antibodies, small organic molecules andsmall inorganic molecules. In a preferred embodiment the test compoundsare peptides. The peptides may be anchored to a solid support byspecific binding to an immobilized antibody. In addition, the testcompounds may be contained in a cellular extract, particularly acellular extract prepared from cells known to express an MrgDpolypeptide, such as dorsal root ganglion cells.

In another aspect, the invention provides a method of indentifying acompound that binds an MrgD polypeptide by contacting an MrgDpolypeptide or fragment with a test compound and a ligand, such as anRFamide peptide, under conditions where binding can occur. Preferablythe MrgD polypeptide is contacted with the RFamide peptide prior tobeing contacted with the test compound. The ability of the test compoundto interfere with biding of the RFamide peptide to the MrgD polypeptideis determined.

In one embodiment the MrgD polypeptide is a native human MrgDpolypeptide, preferably the MrgD polypeptide of SEQ ID NO: 35.

The invention also provides a method of identifying an MrgD agonist thatcan be used to alter sensory perception in a mammal. For example, theagonist may be used to enhance or decreast the preception of pain. AnMrgD polypeptide is expressed in a host cell capable of producing asecond messenger response. In one embodiment the host cell is aeukaryotic cell, preferably a hamster embryonic kidney (HEK) cell, morepreferably an HEK cell that expresses Gα15.

The host cell is contacted with one or more test compounds and thesecond messenger response is measured. Compounds that increase themeasured second messenger response are identified as agonists that canbe used to alter sensory perception in a mammal. In one embodimentmeasuring the second messenger response comprises measuring a change inintercellular calcium concentration. This may be done, for example, byusing a FURA-2 indicator dye. In another embodiment a second messengerresponse is measured by measuring the flow of current across the cellmembrane.

In another aspect, the invention provides a method for identifying anMrgD polypeptide antagonist that is useful in treating impaired sensoryperception in a mammal. In particular the method is useful foridentifying antagonists that can alter the perception of pain.

In one embodiment, an MrgD polypeptide, preferably the MrgD polypeptideof SEQ ID NO: 35, is expressed in a host cell capable of producing asecond messenger response. The host cell is then contacted with anRFamide peptide and one or more test compounds. The second messengerresponse is measured, such as by the methods described above, andcompounds that alter the second messenger response to the RFamidepeptide are identified as agonists that are useful in treating impairedsensory perception, such as pain.

In yet another aspect, the present invention provides a method ofidentifying an anti-MrgD agonist antibody that can be used to alter theperception of pain in a mammal. In one embodiment the anti-MrgD agonistantibody the method is used to identify anti-MrgD agonist antibodiesthat can be used to treat pain in a mammal that is suffering from pain.

In a preferred embodiment, candidate antibodies are prepared thatspecifically bind to an MrgD polypeptide, more preferably to the MrgDpolypeptide of SEQ ID NO: 35. An MrgD polypeptide, preferably the MrgDpolypeptide of SEQ ID NO: 35, is expressed in a host cell known to becapable of producing a second messenger response. The host cell is thencontacted with a candidate antibody and the second messenger response ismeasured. Antibodies that increase the second messenger response areidentified as agonist antibodies that can be used to treat pain in amammal.

The invention also provides a method of treating pain in a mammal,comprising administering to the mammal an MrgD agonist. In oneembodiment, the agonist is an agonist of the human MrgD polypeptide ofSEQ ID NO: 35.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the alignment of a homologous region of the amino acidsequences of SEQ ID NO: 2, 4, 6, 8, 10 and 12, and also of two humanmembers of the mrg family (SEQ ID NOS: 16 and 18).

FIGS. 1B-D indicate that mrgs define a Novel G protein-couple receptorGene Family. Amino acid sequences of eight mouse full-length mrg geneswere aligned using ClustalW. The sequence depicted are: mrg3 (SEQ ID NO:2), mrg4 (SEQ ID NO: 4), mrg5 (SEQ ID NO: 6), mrg8 (SEQ ID NO: 12), mrg9(SEQ ID NO: 21), mrg10 (SEQ ID NO: 23), mrg11 (SEQ ID NO: 25), and mrg12(SEQ ID NO: 27). Identical residues in >50% of the predicted proteinsare darkly shaded; conservative substitutions are highlighted in lightgray. The approximate locations of predicted transmembrane domain 1-7are indicated on top of the sequences as TM1-TM7. The predictedextracellular and cytoplasmic domains are indicated as E1-E7 and C1-C7respectively.

The microscopy images of in situ hybridization in FIG. 2 show thelocalization of antisense staining against a nucleotide of SEQ ID NO: 2(“mrg3”) and of SEQ ID NO: 4 (“mrg4”) in transverse sections of dorsalroot ganglia (DRG) from newborn wild type (WT) and Neurogenin1 nullmutant (Ngn1^(−/−)) mice. White dashed lines outline the DRG and blackdashed lines outline the spinal cord. Note that in the Ngn1^(−/−)mutant, the size of the DRG is severely reduced due to the loss ofnociceptive sensory neurons, identified using three other independentmarkers (trkA; VR-1 and SNS-TTXi (Ma et al., (1999)). mrg3 is expressedin a subset of DRG in WT mice (A) but is absent in the Ngn1^(−/−) DRG(B). mrg4 is expressed in a smaller subset of DRG than that of mrg3 (C).It is also absent in the Ngn1^(−/−) DRG (D). The loss of mrg-expressingneurons in the Ngn1^(−/−) DRG indicates that these neurons are likely tobe nociceptive.

FIG. 2A shows expression of mrgs in subsets of dorsal root ganglia (DRG)neurons. Frozen transverse sections of DRG from wild-type (a-i) andngn1^(−/−) (j) mutant new born mice were annealed with antisensedigoxigenin RNA probes, and hybridization was visualized with analkailine phosphatase-conjugated antibody. Positive signals are shown asdark purple stainings. TrkA is expressed in a large portion of wild-typeDRG neurons (a) but absent in ngn1^(−/−) (data not shown). Each of theeight mrg genes (b-i) is expressed in a small subset of neurons inwild-type DRG in completely absent in ngn1^(−/−) DRG (j and data notshown). Black dash line outlines the ngn1^(−/−) mutant DRG.

FIG. 2B shows that mrgs are expressed by TrkA⁺ nociceptive neurons.Double labeling technique was used to colocalize TrkA (b,e) and mrgs(a,d) in DRG neurons. During the double labeling experiments frozensections of wild-type DRG were undergone in situ hybridizations witheither mrg3 (a-c) or mrg5 (d-f) fluorescein-labeled antisense RNA probesfollowed by anti-TrkA antibody immunostaining. The same two frames (aand b, d and e) were digitally superimposed to reveal the extent ofcolocalization (c, f). The white arrowheads indicate examples of doublepositive cells.

FIG. 2C shows that mrgs and VR1 define two different populations ofnociceptive neurons in DRG. The combination of in situ hybridizationswith either mrg3 or mrg5 fluorescein-labeled antisense RNA probes andanti-VR1 antibody immunostaining demonstrated that neither mrg3 (a-c)nor mrg5 (d-f) were expressed by VR1-positive neurons. In the mergedimages (c,f), there are no colocalizations of VR1 with either mrg3 ormrg5. The white arrowheads are pointed to mrgs-expressing butVR1-negative nociceptive neurons.

FIG. 2D shows that mrgs are expressed by IB4⁺ nociceptive neurons.Double labeling technique was used to colocalize IB4 (b,e) and mrgs(a,d) in DRG neurons. The expressions of mrg3 and mrg5 were visualizedby in situ hybridization as described before. The same DRG sections weresubsequently undergone through FITC-conjugated lectin IB4 binding. Inthe merged images (c,f), there are extensive overlappings between mrgsand IB4 stainings (yellow neurons indicated by arrowheads).

FIG. 3 compares the hydrophobicity plots predicting the transmembraneregions of the amino acid sequence of (A) mrg3 (SEQ ID NO: 2); (B)human1 gene (SEQ ID NO: 15); and (C) human2 gene (SEQ ID NO: 17). Morepositive values indicate hydrophobicity.

FIG. 4 compares the hydrophobicity plots predicting the transmembraneregions of the amino acid sequence of (A) mouse drg12 (SEQ ID NO: 14);(B) human drg12 (SEQ ID NO: 19)

FIG. 5 compares the hydrophobicity plots predicting the transmembraneregions of the amino acid sequence of mrg9 (SEQ ID NO: 21); mrg10 (SEQID NO: 23); mrg11 (SEQ ID NO: 25) and mrg12 (SEQ ID NO: 27).

FIG. 6A is an alignment of the amino acid sequences of MRGA1-A8 deducedfrom nucleotide sequences of cDNA and BAC clones from strain C57BL/6Jmice. MRGA1-A8 are depicted as mrg3 (MrgA1, SEQ ID NO: 2), mrg4 (MrgA2,SEQ ID NO: 4), mrg5 (MrgA3, SEQ ID NO: 6), mrg8 (MrgA4, SEQ ID NO: 12),mrg9 (MrgA5, SEQ ID NO: 21), mrg10 (MrgA6, SEQ ID NO: 23), mrg11 (MrgA7,SEQ ID NO: 25), and mrg12 (MrgA8, SEQ ID NO: 27). The predictedlocations of the transmembrane (TM1-TM7), extracellular (E1-E4), andcytoplasmic (C1-C4) domains are indicated above the aligned sequences.

FIG. 6B depicts a phylogenetic analysis of MRG family members identifiedfrom database searches. The protein sequences of all MRGs were alignedusing CLUSTALW (Thompson et al. Nucleic Acids Res 22: 4673-80 (1994)).The dendrogram was generated with the PHYLUP software package using theNeighbor-Joining method and 1,000 bootstrap trials. The horizontallength of the branches is proportional to the number of amino acidchanges. Vertical distances are arbitrary. Mouse (m)Mrg genes withretrotransposon sequences ˜650 nt 3′ of their stop codon are highlighted(L1). All genes that are predicted to encode pseudogenes are indicatedwith the psi (Ψ) symbol.

FIG. 6C shows the chromosomal organization of one mouse Mrg clusterdeduced from analysis of overlapping BAC clones. The cluster containsfour intact ORFs and three pseudogenes.

FIG. 7A shows the distribution of nociceptive sensory neurons in apostnatal day 0 (P0) DRG as revealed by expression of the NGF receptortrkA. This population is selectively eliminated in Ngn1^(−/−) mutants(Ma et al. Genes & Dev. 13: 1717-1728 (1999)).

FIG. 7B shows in situ hybridization with cRNA probes detecting MrgA1.MrgA1 is expressed in a pattern similar to that of trkA⁺ neurons on anadjacent section shown in FIG. 7A.

FIG. 7C-I shows in situ hybridization with cRNA probes detectingMrgA2-MrgA8.

FIG. 7J shows that MrgA1 expression is eliminated in Ngn1^(−/−) mice, asis expression of other MrgA genes (not shown). Remaining DRG neurons arepresent in the area delimited by the dotted line, and can be visualizedby expression of generic neuronal markers.

FIG. 8 shows that expression of MrgAs is restricted to non-peptidergicnociceptors that project to inner lamina II. Shown are confocalmicroscopic images of in situ hybridizations using the Mrg probesindicated, combined with fluorescent antibody detection of trkA (A-D),substance P (I-L), CGRP (M-P), VR1 (Q-T) or staining with fluorescentisolectin IB4 (IB4; E-H). MrgA⁺ or MrgD⁺ cells co-express trkA and IB4(A-H, arrowheads), but most do not express subP, CGRP or VR1 (I-T,arrowheads; arrows in I, M indicate a minor subset of MrgA1⁺ neuronsthat co-express SubP and CGRP).

FIG. 9 is a schematic illustration of the restriction of MrgA (and MrgD)expression to non-peptidergic, IB4⁺, VR1⁻ sensory neurons that projectto lamina IIi (Snider and McMahon Neuron 20: 629-32 (1998)).Post-synaptic neurons in lamina IIi express PKCγ.

FIG. 10 shows that individual sensory neurons co-express multiple MrgAs.(A-C) double label in situ hybridization with MrgA1 (A) and A3 (B).(D-F) double labeling with MrgA1 (D) and MrgA4 (E). In both cases, cellsexpressing MrgA3 or A4 are a subset of those expressing MrgA1 (C, F,arrowheads). Arrows in (F) indicate intranuclear dots of MrgA4expression which may represent sites of transcription. (G-I) Doublelabel in situ with MrgA1 and MrgD. Some overlap overlap between the twopopulations is seen (I, arrowhead), while most cells express onereceptor but not the other (I, arrows). Approximately 15% of cellsexpressing either MrgA1 or MrgD co-express both genes. Vertical bars tothe right of panels (C, F, I) represent a z-series viewed along they-axis, horizontal bars below the panels a z-series viewed along thex-axis. (J, K) comparison of in situ hybridization signals obtainedusing a single MrgA probe (J) and a mixture of 7 MrgA probes (K).Approximately 1% of neurons were labeled by the MrgA4 probe, while ˜4.5%were labeled by the mixed probe. The sum of the percentage of neuronslabeled by the individual MrgA2-8 probes is ˜6.6%, suggesting that thereis partial overlap within this population. (L) Venn diagram illustratingcombinations of gene expression revealed by in situ analysis. Thedrawing is a conservative estimate of the number of subsets, since we donot yet know, for example, whether MrgAs2-8 partially overlap with MrgD.The sizes of the circles are not proportional.

FIG. 11 shows elevated intracellular free Ca⁺⁺ elicited by FLRF in HEKcells expressing MRGA1. (A, B) and (E, F) illustrate Fura-2 fluorescenceat 340 nm (A, E) and 380 nm (B, F) in HEK-Gα₁₅ cells expressing anMRGA1-GFP fusion protein (A-D) or GFP alone (E-H). The images were taken2 minutes after the addition of 1 μM of FLRFamide. The peri-nuclear,punctate distribution of MRGA1-GFP revealed by intrinsic GFPfluorescence (D, arrowheads) is characteristic of the ER-Golgi network,indicating membrane integration and intracellular transport of thereceptors. In contrast, the control GFP protein is cytoplasmic (H). Theintracellular Ca²⁺ ([Ca²⁺]_(i)) release was determined from the FURA-2340 nM/380 nM emission ratio (C, G). Note that MRGA1-expressing cells(but not surrounding untransfected cells) show an elevated ratio ofFura-2 fluorescence at 340/380 nm (C, arrowheads), indicating anincrease in [Ca²⁺]_(i). In contrast, no such elevation is observed incontrol GFP-expressing cells (G). The elevated 340/380 fluorescence seenin MRGA1-expressing cells was dependent on the addition of ligand (notshown).

FIG. 12A shows activation of MRGA receptors expressed in heterologouscells by neuropeptide ligands. HEK-Gα₁₅ cells (Offermanns and Simon. JBiol Chem 270: 15175-80 (1995)) expressing MRGA1 were tested with theindicated ligands at a concentration of 1 μM. The data indicate the meanpercentages of GFP-positive (i.e., transfected) cells showing calciumresponses. None of the agonists indicated showed any responses throughendogenous receptors in untransfected cells. Note that the RFamideneuropeptides FMRF, FLRF and NPFF, as well as NPY, ACTH, CGRP-I and -IIand somatostatin (SST) produced the strongest responses.

FIG. 12B shows the ligand selectivity of MRGA1 expressed in HEK cellslacking Gα₁₅. The cells were exposed to ligands at a concentration of 1μM as in (A).

FIG. 12C shows the ligand selectivity of MRGA4. The data presented inFIGS. 12B and 12C indicate that the responses to the most effectiveligands do not depend on the presence of Gα₁₅. Note thatMRGA1-expressing cells respond to FLRF and NPFF but not to NPAF, whileconversely MRGA4-expressing cells respond to NPAF but not NPFF or FLRF

FIG. 12D shows dose-response curves for MRGA1 expressed in HEK-Gα₁₅cells to selected RFamide neuropeptides. Each data point represents themean±S.E.M. of at least 3 independent determinations; at least 20 GFP⁺cells were analyzed for each determination. Responses at each ligandconcentration were normalized to the maximal response subsequently shownby the same cells to a 5 μM concentration of FLRF. MRGA1 (D) showshighest sensitivity to FLRF (squares, EC₅₀≈20 nM) and lower sensitivityto NPFF (circles, EC₅₀≈200 nM).

FIG. 12E shows dose-response curves for MRGA4 expressed in HEK-Gα₁₅cells to selected RFamide neuropeptides. Each data point represents themean±S.E.M. of at least 3 independent determinations; at least 20 GFP⁺cells were analyzed for each determination. Responses at each ligandconcentration were normalized to the maximal response subsequently shownby the same cells to a 5 μM concentration of NPAF. MRGA4 ispreferentially activated by NPAF (triangles, EC₅₀≈60 nM).

FIG. 12F shows dose-response curves for MAS1 expressed in HEK-Gα₁₅ cellsto selected RFamide neuropeptides. Each data point represents themean±S.E.M. of at least 3 independent determinations; at least 20 GFP⁺cells were analyzed for each determination. Responses at each ligandconcentration were normalized to the maximal response subsequently shownby the same cells to a 5 μM concentration of NPFF. MAS1, like MRGA1, isactivated by NPFF with similar efficacy (EC₅₀≈400 nM), but is not aswell activated by FLRF (squares).

FIG. 13 depicts the expression pattern of mMrgB1 in a sagital section ofa newborn mouse. The staining pattern indicates that the mMrgB1 gene isexpressed in the scattered cells in the epidermal layer of the skin, inthe spleen and in the submandibular gland.

FIG. 14 is a higher magnification of the mMrgB1 expression in the spleenand skin depicted in FIG. 13.

FIG. 15 shows the expression of mMrgD in adult dorsal root ganglia.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. General Description

As described above, the present invention is based on the discovery ofnew genes that are expressed in the DRG of normal mice but not in Ngn1null mutant mice. One of the novel gene families isolated from thescreen encodes a receptor protein with 7 transmembrane segments, acharacteristic of G protein-coupled receptors. This novel 7transmembrane receptor is most closely related to the oncogene mas, andtherefore was provisionally named mas-related gene-3 (mrg3). mrg3 is nowknown as MrgA1, and the terms are used interchangeably herein. Almost 50members of the Mas-related gene (Mrg) family have been identified, manyof which are specifically expressed in non-peptidergic nociceptors.Large families of G protein-coupled receptors are also expressed inother classes of sensory neurons, such as olfactory and gustatoryneurons.

The murine Mrg family of GPCRs contains three major subfamilies (MrgA, Band C), each consisting of more than 10 highly duplicated genes, as wellas several single-copy genes such as Mas1, Rta, MrgD and MrgE (FIG. 6B).The MrgA subfamily consists of at least twenty members in mice: MrgA1(SEQ ID NO: 2); MrgA2 (SEQ ID NO: 4); MrgA3 (SEQ ID NO: 6); MrgA4 (SEQID NO: 11); MrgA5 (SEQ ID NO: 21); MrgA6 (SEQ ID NO: 23); MrgA7 (SEQ IDNO: 25); MrgA8 (SEQ ID NO: 27); MrgA9 (SEQ ID NO: 53); MrgA10 (SEQ IDNO: 55); MrgA11 (SEQ ID NO: 57); MrgA12 (SEQ ID NO: 59); MrgA13 (SEQ IDNO: 61); MrgA14 (SEQ ID NO: 63); MrgA15 (SEQ ID NO: 65); MrgA16 (SEQ IDNO: 67); MrgA17 (SEQ ID NO: 69); MrgA18 (SEQ ID NO: 71); MrgA19 (SEQ IDNO: 73); MrgA20 (SEQ ID NO: 75). Four human sequences that are mostcloses related to the MrgA subfamily have also been identified: MrgX1(SEQ ID NO: 16); MrgX2 (SEQ ID NO: 18); MrgX3 (SEQ ID NO: 31); and MrgX4(SEQ ID NO: 33).

The MrgB subfamily consists of at least twelve members in mice: MrgB1(SEQ ID NO: 39); MrgB2 (SEQ ID NO: 41); MrgB3 (SEQ ID NO: 43); MrgB4(SEQ ID NO: 45); MrgB5 (SEQ ID NO: 47); MrgB6 (SEQ ID NO: 77); MrgB7(SEQ ID NO: 79); MrgB8 (SEQ ID NO: 81); MrgB9 (SEQ ID NO: 83); MrgB10(SEQ ID NO: 85); MrgB11 (SEQ ID NO: 87); and MrgB12 (SEQ ID NO: 89).

Ten members of the MrgC subfamily have been identified in mice: MrgC1(SEQ ID NO: 91); MrgC2 (SEQ ID NO: 93); MrgC3 (SEQ ID NO: 95); MrgC4(SEQ ID NO: 97); MrgC5 (SEQ ID NO: 99); MrgC6 (SEQ ID NO: 101); MrgC7(SEQ ID NO: 103); MrgC8 (SEQ ID NO: 105); MrgC9 (SEQ ID NO: 107); andMrgC10 (SEQ ID NO: 109).

A single member of the MrgD subfamily has been identified in mice, mMrgD(SEQ ID NO: 49) and its ortholog identified in humans, hMrgD (SEQ ID NO:35). Similarly, a single member of the MrgE subfamily has beenidentified in mice, mMrgE (SEQ ID NO: 51) and humans, hMrgE (SEQ ID NO:37).

As is the case in other GPCR subfamilies, a number of the Mrgs appear tobe pseudogenes, including all members of the MrgC subfamily. Thepresence of L1 retrotransposon elements near several Mrg genes raisesthe possibility that pseudogene expansion may have been driven byL1-mediated transduction (Goodier et al. Hum Mol Genet 9: 653-7 (2000)).

In contrast to the murine MrgA and B subfamilies, which together containalmost 40 intact coding sequences, only four intact human MrgX sequenceswere identified. The remaining 10 human Mrg sequences appear to bepseudogenes. Inclusion of other related receptors such as hMrgD andhMas1 brings the total number of intact human coding sequences in thisfamily to nine (FIG. 6B).

Prior to the present invention, the primary nociceptive sensory neuronswere thought not to specifically discriminate among different chemicalstimuli, but rather to detect noxious stimuli of various modalities byvirtue of broadly tuned receptors such as VR1 (Tominaga et al. Neuron21: 531-43 (1998)). The expression of Mrgs reveals an unexpected degreeof molecular diversification among nociceptive sensory neurons.Approximately 13-14% of sensory neurons express MrgA1, while 17-18%express MrgD and the overlap between these two populations is only 15%.The MrgA1⁺ population seems to include most or all neurons expressingMrgA2-8. However, these latter MrgA genes are not all expressed in thesame neurons. Thus the 8 MrgA genes and MrgD define at least 6 differentneuronal subpopulations, and the remaining 16 MrgA genes add evengreater diversity.

It is striking that both MrgA and D are expressed in IB4⁺, VR1⁻ sensoryneurons. IB4⁺ neurons are known to project to lamina IIi (Snider andMcMahon Neuron 20: 629-32 (1998)), which has been implicated in chronicpain, such as that accompanying nerve injury (Malmberg et al. Science278: 279-83 (1997)). VR1 is activated both by thermal stimuli andchemical stimuli such as capsaicin (Caterina et al. Nature 389: 816-824(1997); Tominaga et al. Neuron 21: 531-43 (1998)), but VR1⁺ neurons aredispensable for the detection of noxious mechanical stimuli (Caterina etal. Science 288: 306-13 (2000)). This indicates that one of thefunctions of MrgA⁺ neurons is the detection of noxious mechanicalstimuli accompanying neuropathic or inflammatory pain.

The existence of a family of putative G protein-coupled receptorsspecifically expressed in nociceptive sensory neurons suggests thatthese molecules are primary mediators or modulators of pain sensation.It is therefore of great interest to identify ligands, both endogenousand synthetic, that modulate the activity of these receptors, for themanagement of chronic intractable pain. Indeed, ligand screens inheterologous cell expression systems indicate that these receptors caninteract with RF-amide neuropeptides of which the prototypic member isthe molluscan cardioexcitatory peptide FMRF-amide (Price and GreenbergScience 197: 670-671 (1977)). Mammalian RF-amide peptides include NPFFand NPAF, which are derived from a common pro-peptide precursorexpressed in neurons of laminae I and II of the dorsal spinal cord(Vilim et al. Mol Pharmacol 55: 804-11 (1999)). The expression of thisneuropeptide FF precursor in the synaptic termination zone ofMrg-expressing sensory neurons, the ability of NPAF and NPFF to activatethese receptors in functional assays, and the presence of binding sitesfor such peptides on primary sensory afferents in the dorsal horn(Gouarderes et al. Synapse 35: 45-52 (2000)), together indicate thatthese neuropeptides are ligands for Mrg receptors in vivo. Asintrathecal injection of NPFF/NPAF peptides produces long-lastingantinociceptive effects in several chronic pain models (reviewed inPanula et al. Brain Res 848: 191-6 (1999)), including neuropathic pain(Xu et al. Peptides 20: 1071-7 (1999)), these data further indicate thatMrgs are directly involved in the modulation of pain.

One possibility for the extent of diversity among Mrgs expressed bymurine nociceptors is that different Mrgs are expressed by sensoryneurons that innervate different peripheral targets, such as gut, skin,hair follicles, blood vessels, bones and muscle. These targets maysecrete different ligands for different Mrgs. Another possibility isthat neurons expressing different Mrgs respond to a common modulator ofperipheral nociceptor sensitivity, but with different affinities. Such amechanism could, for example, provide a gradual restoration of normalsensitivities among the population of nociceptors during wound healing,as the concentration of such modulators gradually returned to baseline.Such a graded response might be coupled to, or even determine theactivation thresholds of different subsets of nociceptors. Another novelgene family isolated in this screen, drg-12 encodes a protein with twoputative transmembrane segments. Drg12 was identified from both mice(SEQ ID NO: 14) and in humans (SEQ ID NO: 29). In situ hybridizationindicates that, like the mrg genes, this gene is also specificallyexpressed in a subset of DRG sensory neurons. As it is a membraneprotein it may also be involved in signaling by these neurons. Althoughthere are no obvious homologies between this protein and other knownproteins, it is noteworthy that two purinergic receptors specificallyexpressed in nociceptive sensory neurons (P₂X₂ and P₂X₃) have a similarbipartite transmembrane topology. Therefore it is likely that the familydrg-12 also encodes a receptor or ion channel involved in nociceptivesensory transduction or its modulation.

The proteins of the invention can serve as therapeutics and as a targetfor agents that modulate their expression or activity, for example inthe treatment of chronic intractable pain and neuropathic pain. Forexample, agents may be identified which modulate biological processesassociated with nociception such as the reception, transduction andtransmission of pain signals.

II. Specific Embodiments

A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

As used herein, the “protein” or “polypeptide” refers, in part, to aprotein that has the amino acid sequence depicted in SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109.The terms also refer to naturally occurring allelic variants andproteins that have a slightly different amino acid sequence than thosespecifically recited above. Allelic variants, though possessing aslightly different amino acid sequence than those recited above, willstill have the same or similar biological functions associated with theprotein.

Identity or homology with respect to amino acid sequences is definedherein as the percentage of amino acid residues in the candidatesequence that are identical with the known peptides, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent homology, and not considering any conservative substitutions aspart of the sequence identity (see section B for the relevantparameters). Fusion proteins, or N-terminal, C-terminal or internalextensions, deletions, or insertions into the peptide sequence shall notbe construed as affecting homology.

Proteins can be aligned using CLUSTALW (Thompson et al. Nucleic AcidsRes 22:4673-80 (1994)) and homology or identity at the nucleotide oramino acid sequence level may be determined by BLAST (Basic LocalAlignment Search Tool) analysis using the algorithm employed by theprograms blastp, blastn, blastx, tblastn and tblastx (Karlin, et al.Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, S. F. J.Mol. Evol. 36: 290-300 (1993), fully incorporated by reference) whichare tailored for sequence similarity searching. The approach used by theBLAST program is to first consider similar segments between a querysequence and a database sequence, then to evaluate the statisticalsignificance of all matches that are identified and finally to summarizeonly those matches which satisfy a preselected threshold ofsignificance. For a discussion of basic issues in similarity searchingof sequence databases, see Altschul et al. (Nature Genetics 6: 119-129(1994)) which is fully incorporated by reference. The search parametersfor histogram, descriptions, alignments, expect (i.e., the statisticalsignificance threshold for reporting matches against databasesequences), cutoff, matrix and filter are at the default settings. Thedefault scoring matrix used by blastp, blastx, tblastn, and tblastx isthe BLOSUM62 matrix (Henikoff, et al. Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992), fully incorporated by reference). For blastn, thescoring matrix is set by the ratios of M (i.e., the reward score for apair of matching residues) to N (i.e., the penalty score for mismatchingresidues), wherein the default values for M and N are 5 and -4,respectively. Four blastn parameters were adjusted as follows: Q=10 (gapcreation penalty); R=10 (gap extension penalty); wink=1 (generates wordhits at every winkth position along the query); and gapw=16 (sets thewindow width within which gapped alignments are generated). Theequivalent Blastp parameter settings were Q=9; R=2; wink=1; and gapw=32.A Bestfit comparison between sequences, available in the GCG packageversion 10.0, uses DNA parameters GAP=50 (gap creation penalty) andLEN=3 (gap extension penalty) and the equivalent settings in proteincomparisons are GAP=8 and LEN=2.

“Variants” are biologically active polypeptides having an amino acidsequence which differs from the sequence of a native sequencepolypeptide of the present invention, such as that shown in FIG. 1A formrg3 (SEQ ID NO: 2), by virtue of an insertion, deletion, modificationand/or substitution of one or more amino acid residues within the nativesequence. Variants include peptide fragments of at least 5 amino acids,preferably at least 10 amino acids, more preferably at least 15 aminoacids, even more preferably at least 20 amino acids that retain abiological activity of the corresponding native sequence polypeptide.Variants also include polypeptides wherein one or more amino acidresidues are added at the N- or C-terminus of, or within, a nativesequence. Further, variants also include polypeptides where a number ofamino acid residues are deleted and optionally substituted by one ormore different amino acid residues.

As used herein, a “conservative variant” refers to alterations in theamino acid sequence that do not adversely affect the biologicalfunctions of the protein. A substitution, insertion or deletion is saidto adversely affect the protein when the altered sequence prevents ordisrupts a biological function associated with the protein. For example,the overall charge, structure or hydrophobic/hydrophilic properties ofthe protein can be altered without adversely affecting a biologicalactivity. Accordingly, the amino acid sequence can be altered, forexample to render the peptide more hydrophobic or hydrophilic, withoutadversely affecting the biological activities of the protein.

As used herein, the “family of proteins” related to the amino acidsequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107 and 109 includes proteins that have been isolatedfrom the dorsal root ganglia of organisms in addition to mice andhumans. The methods used to identify and isolate other members of thefamily of proteins related to these proteins, such as the disclosedmouse and human proteins, are described below.

Unless indicated otherwise, the term “Mrg” when used herein refers toany one or more of the mammalian mas-related gene (Mrg) receptors (i.e.MrgA1-8, MrgB, MrgC, MrgD, MrgE, MrgX1-4 and any other members of themas-related gene (Mrg) family now known or identified in the future),including native sequence mammalian, such as murine or human, Mrgreceptors, Mrg receptor variants; Mrg receptor extracellular domain; andchimeric Mrg receptors (each of which is defined herein). The termspecifically includes native sequence murine Mrg receptors of the MrgAfamily, such as SEQ ID NOs: 2, 4, 6 12, 21, 23, 25, 27, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, and 75; native sequence murine Mrg receptorsof the MrgB family, such as SEQ ID NOs: 39, 41, 43, 45, 47, 77, 79, 81,83, 85, 87, and 89; native sequence murine Mrg receptors of the MrgCfamily, such as SEQ ID NOs: 91, 93, 95, 97, 99, 101, 103, 105, 107 and109; native sequence murine Mrg receptors of the MrgD family, such asSEQ ID NO: 49; native sequence murine Mrg receptors of the MrgE family,such as SEQ ID NO: 51; their human homologues, and the native sequencehuman Mrg receptors termed “MrgX” of SEQ ID NOs: 16, 18, 31 and 33.

The terms “mas-related gene”, “mrg” and “Mrg” are used interchangeablyherein. Further, the terms mrg3, MrgA1 and mMrgA1 are usedinterchangeably, as are the terms mrg4, MrgA2 and mMrgA2, the termsmrg5, MrgA3 and mMrgA3, the terms mrg8, MrgA4 and mMrgA4, the termsmrg9, MrgA5 and mMrgA5, the terms mrg10, MrgA6 and mMrgA6, the termsmrg11, MrgA7 and mMrgA7, the terms mrg12, MrgA8 and mMrgA8, the termshuman1, MrgX1 and hMrgX1, the terms human2, MrgX2 and hMrgX2, the termshuman 4, MrgX3 and hMrgX3, and the terms human5, MrgX4 and hMrgX4. Theseterms all refer to native sequence Mrg proteins as described herein aswell as functional derivatives, including amino acid sequence variantsthereof.

A “native” or “native sequence” Mrg or drg-12 receptor has the aminoacid sequence of a naturally occurring Mrg or drg-12 receptor in anymammalian species (including humans), irrespective of its mode ofpreparation. Accordingly, a native or native sequence Mrg or drg-12receptor may be isolated from nature, produced by techniques ofrecombinant DNA technology, chemically synthesized, or produced by anycombinations of these or similar methods. Native Mrg and drg-12receptors specifically include polypeptides having the amino acidsequence of naturally occurring allelic variants, isoforms or splicedvariants of these receptors, known in the art or hereinafter discovered.

The “extracellular domain” (ECD) is a form of the Mrg or drg-12 receptorwhich is essentially free of the transmembrane and cytoplasmic domains,i.e., has less than 1% of such domains, preferably 0.5 to 0% of suchdomains, and more preferably 0.1 to 0% of such domains. Ordinarily, theECD will have an amino acid sequence having at least about 60% aminoacid sequence identity with the amino acid sequence of one or more ofthe ECDs of a native Mrg or drg-12 protein, for example as indicated inFIGS. 1B-D for mrg3 (E1, E2 etc. . . . ), preferably at least about 65%,more preferably at least about 75%, even more preferably at least about80%, even more preferably at least about 90%, with increasing preferenceof 95%, to at least 99% amino acid sequence identity, and finally to100% identity, and thus includes polypeptide variants as defined below.

The first predicted extracellular domain (ECD1) comprises approximatelyamino acids 1 to 21 for MrgA1, 1 to 21 for MrgA2, 1 to 21 for MrgA3, 1to 21 for MrgA4, 1 to 3 for MrgA5, 1 to 17 for MrgA6, 1 to 21 for MrgA7and 1 to 21 for MrgA8. The second predicted extracellular domain (ECD2)comprises approximately amino acids 70 to 87 for MrgA1, 70 to 88 forMrgA2, 70 to 88 for MrgA3, 70 to 88 for MrgA4, 52 to 70 for MrgA5, 66 to84 for MrgA6, 70 to 88 for MrgA7 and 70 to 88 for MrgA8. The thirdpredicted extracellular domain (ECD3) comprises approximately aminoacids 149 to 160 for MrgA1, 150 to 161 for MrgA2, 150 to 161 for MrgA3,150 to 161 for MrgA4, 132 to 144 for MrgA5, 146 to 157 for MrgA6, 150 to161 for MrgA7 and 150 to 161 for MrgA8. The fourth predictedextracellular domain (ECD4) comprises approximately amino acids 222 to2244 for MrgA1, 223 to 245 for MrgA2, 223 to 242 for MrgA3, 223 to 245for MrgA4, 205 to 225 for MrgA5, 219 to 241 for MrgA6, 223 to 245 forMrgA7 and 223 to 245 for MrgA8.

The term “drg-12” when used herein refers to any one or more of themammalian drg-12 receptors now known or identified in the future,including native sequence mammalian, such as murine or human, drg-12receptors, drg-12 receptor variants; drg-12 receptor extracellulardomain; and chimeric drg-12 receptors (each of which is defined herein).The term specifically includes native sequence murine drg-12 receptor,such as SEQ ID NO: 14, and any human homologues, such as human drg-12(SEQ ID NO: 29).

As used herein, “nucleic acid” is defined as RNA or DNA that encodes aprotein or peptide as defined above, is complementary to a nucleic acidsequence encoding such peptides, hybridizes to such a nucleic acid andremains stably bound to it under appropriate stringency conditions,exhibits at least about 50%, 60%, 70%, 75%, 85%, 90% or 95% nucleotidesequence identity across the open reading frame, or encodes apolypeptide sharing at least about 50%, 60%, 70% or 75% sequenceidentity, preferably at least about 80%, and more preferably at leastabout 85%, and even more preferably at least about 90 or 95% or moreidentity with the peptide sequences. Specifically contemplated aregenomic DNA, cDNA, mRNA and antisense molecules, as well as nucleicacids based on alternative backbones or including alternative baseswhether derived from natural sources or synthesized. Such hybridizing orcomplementary nucleic acids, however, are defined further as being noveland unobvious over any prior art nucleic acid including that whichencodes, hybridizes under appropriate stringency conditions, or iscomplementary to nucleic acid encoding a protein according to thepresent invention.

As used herein, the terms nucleic acid, polynucleotide and nucleotideare interchangeable and refer to any nucleic acid, whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sultone linkages, andcombinations of such linkages.

The terms nucleic acid, polynucleotide and nucleotide also specificallyinclude nucleic acids composed of bases other than the five biologicallyoccurring bases (adenine, guanine, thymine, cytosine and uracil). Forexample, a polynucleotide of the invention might contain at least onemodified base moiety which is selected from the group including but notlimited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyl-uracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5N-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

Furthermore, a polynucleotide used in the invention may comprise atleast one modified sugar moiety selected from the group including butnot limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

“Stringent conditions” are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% SDS at 50° C., or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC and 0.1% SDS. A skilled artisan can readily determineand vary the stringency conditions appropriately to obtain a clear anddetectable hybridization signal.

As used herein, a nucleic acid molecule is said to be “isolated” whenthe nucleic acid molecule is substantially separated from contaminantnucleic acid molecules encoding other polypeptides.

As used herein, a fragment of an encoding nucleic acid molecule refersto a small portion of the entire protein coding sequence. The size ofthe fragment will be determined by the intended use. For example, if thefragment is chosen so as to encode an active portion of the protein, thefragment will need to be large enough to encode the functional region(s)of the protein. For instance, fragments which encode peptidescorresponding to predicted antigenic regions may be prepared (see FIGS.3 and 4). If the fragment is to be used as a nucleic acid probe or PCRprimer, then the fragment length is chosen so as to obtain a relativelysmall number of false positives during probing/priming (see thediscussion in Section H).

Highly related gene homologs are polynucleotides encoding proteins thathave at least about 60% amino acid sequence identity with the amino acidsequence of a naturally occurring native sequence polynucleotide of theinvention, such as MrgA1 (SEQ ID NO: 2), preferably at least about 65%,70%, 75%, 80%, with increasing preference of at least about 85% to atleast about 99% amino acid sequence identity, in 1% increments.

The term “mammal” is defined as an individual belonging to the classMammalia and includes, without limitation, humans, domestic and farmanimals, and zoo, sports, or pet animals, such as sheep, dogs, horses,cats or cows. Preferably, the mammal herein is human.

“Functional derivatives” include amino acid sequence variants, andcovalent derivatives of the native polypeptides as long as they retain aqualitative biological activity of the corresponding native polypeptide.

By “Mrg ligand” is meant a molecule which specifically binds to andpreferably activates an Mrg receptor. Examples of Mrg ligands include,but are not limited to RF-amide neuropeptides, such as FMRF, FLRF, NPAF,NPFF, and RFRP-1 for MrgA receptors, such as MrgA1. The ability of amolecule to bind to Mrg can be determined, for example, by the abilityof the putative ligand to bind to membrane fractions prepared from cellsexpressing Mrg.

Similarly, a drg-12 ligand is a molecule which specifically binds to andpreferably activates a drg-12 receptor.

A “chimeric” molecule is a polypeptide comprising a full-lengthpolypeptide of the present invention, a variant, or one or more domainsof a polypeptide of the present invention fused or bonded to aheterologous polypeptide. The chimeric molecule will generally share atleast one biological property in common with a naturally occurringnative sequence polypeptide. An example of a chimeric molecule is onethat is epitope tagged for purification purposes. Another chimericmolecule is an immunoadhesin.

The term “epitope-tagged” when used herein refers to a chimericpolypeptide comprising Mrg or drg-12 fused to a “tag polypeptide”. Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made, yet is short enough such that it does notinterfere with the biological activity of the Mrg or drg-12. The tagpolypeptide preferably is fairly unique so that the antibody against itdoes not substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8-50 amino acid residues (preferably between about 9-30residues). Preferred are poly-histidine sequences, which bind nickel,allowing isolation of the tagged protein by Ni-NTA chromatography asdescribed (See, e.g., Lindsay et al. Neuron 17:571-574 (1996)).

“Agonists” are molecules or compounds that stimulate one or more of thebiological properties of a polypeptide of the present invention. Thesemay include, but are not limited to, small organic and inorganicmolecules, peptides, peptide mimetics and agonist antibodies.

The term “antagonist” is used in the broadest sense and refers to anymolecule or compound that blocks, inhibits or neutralizes, eitherpartially or fully, a biological activity mediated by a receptor of thepresent invention by preventing the binding of an agonist. Antagonistsmay include, but are not limited to, small organic and inorganicmolecules, peptides, peptide mimetics and neutralizing antibodies.

The proteins of the present invention are preferably in isolated form.As used herein, a protein is said to be isolated when physical,mechanical or chemical methods are employed to remove the protein fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated protein. In some instances, isolated proteins of theinvention will have been separated or purified from many cellularconstituents, but will still be associated with cellular membranefragments or membrane constituents.

Thus, “isolated Mrg” and “isolated drg-12” means Mrg or drg-12polypeptide, respectively, that has been purified from a protein sourceor has been prepared by recombinant or synthetic methods and purified.Purified Mrg or drg-12 is substantially free of other polypeptides orpeptides. “Substantially free” here means less than about 5%, preferablyless than about 2%, more preferably less than about 1%, even morepreferably less than about 0.5%, most preferably less than about 0.1%contamination with other source proteins.

“Essentially pure” protein means a composition comprising at least about90% by weight of the protein, based on total weight of the composition,preferably at least about 95% by weight, more preferably at least about90% by weight, even more preferably at least about 95% by weight.“Essentially homogeneous” protein means a composition comprising atleast about 99% by weight of protein, based on total weight of thecomposition.

“Biological property” is a biological or immunological activity, wherebiological activity refer to a biological function (either inhibitory orstimulatory) caused by a native sequence or variant polypeptide moleculeherein, other than the ability to induce the production of an antibodyagainst an epitope within such polypeptide, where the latter property isreferred to as immunological activity. Biological propertiesspecifically include the ability to bind a naturally occurring ligand ofthe receptor molecules herein, preferably specific binding, and evenmore preferably specific binding with high affinity.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins, composed of two identical light (L)chains and two identical heavy (H) chains. Each light chain is linked toa heavy chain by one covalent disulfide bond. while The number ofdisulfide linkages varies among the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intra-chain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end. The constant domain of the light chain isaligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “antibody” herein is used in the broadest sense andspecifically covers human, non-human (e.g. murine) and humanizedmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multi-specific antibodies (e.g., bispecificantibodies), and antibody fragments so long as they exhibit the desiredbiological activity.

“Antibody fragments” comprise a portion of a full-length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmulti-specific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of antibodies wherein the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific and are directed against asingle antigenic site. In addition, monoclonal antibodies may be made byany method known in the art. For example, the monoclonal antibodies tobe used in accordance with the present invention may be made by thehybridoma method first described by Kohler et al., Nature 256:495(1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567). The “monoclonal antibodies” may also be isolated fromphage antibody libraries using the techniques described in Clackson etal., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass. Fragments of chimeric antibodies are also includedprovided they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855(1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are antibodiesthat contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies are generally human immunoglobulins in whichhypervariable region residues are replaced by hypervariable regionresidues from a non-human species such as mouse, rat, rabbit ornon-human primate having the desired specificity, affinity, andcapacity. Framework region (FR) residues of the human immunoglobulin maybe replaced by corresponding non-human residues. In addition, humanizedantibodies may comprise residues that are not found in either therecipient antibody or in the donor antibody. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thehypervariable regions correspond to those of a non-human immunoglobulinand all or substantially all of the FRs are those of a humanimmunoglobulin sequence. The humanized antibody optionally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “epitope” is used to refer to binding sites for (monoclonal orpolyclonal) antibodies on protein antigens.

By “agonist antibody” is meant an antibody which is a ligand for areceptor of the invention and thus, able to activate and/or stimulateone or more of the effector functions of native sequence Mrg or drg-12.

By “neutralizing antibody” is meant an antibody molecule as hereindefined which is able to block or significantly reduce an effectorfunction of a polypeptide of the invention. For example, a neutralizingantibody may inhibit or reduce Mrg or drg-12 activation by a knownligand.

The term “Mrg immunoadhesin” refers to a chimeric molecule thatcomprises at least a portion of an Mrg or drg-12 molecule (native orvariant) and an immunoglobulin sequence. The immunoglobulin sequencepreferably, but not necessarily, is an immunoglobulin constant domain.Immunoadhesins can possess many of the properties of human antibodies.Since immunoadhesins can be constructed from a human protein sequencewith a desired specificity linked to an appropriate human immunoglobulinhinge and constant domain (Fc) sequence, the binding specificity ofinterest can be achieved using entirely human components. Suchimmunoadhesins are minimally immunogenic to the patient, and are safefor chronic or repeated use. If the two arms of the immunoadhesinstructure have different specificities, the immunoadhesin is called a“bispecific immunoadhesin” by analogy to bispecific antibodies.

As used herein, “treatment” is a clinical intervention made in responseto a disease, disorder or physiological condition manifested by apatient. The aim of treatment includes the alleviation or prevention ofsymptoms, slowing or stopping the progression or worsening of a disease,disorder, or condition and the remission of the disease, disorder orcondition. “Treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already affected by a disease or disorder or undesiredphysiological condition as well as those in which the disease ordisorder or undesired physiological condition is to be prevented.Specifically, treatment may alleviate pain, including pain resultingfrom an existing condition or disorder, or to prevent pain in situationswhere pain is likely to be experienced.

In the methods of the present invention, the term “control” andgrammatical variants thereof, are used to refer to the prevention,partial or complete inhibition, reduction, delay or slowing down of anunwanted event, such as the presence or onset of pain.

The term “effective amount” refers to an amount sufficient to effectbeneficial or desirable clinical results. An effective amount of anagonist or antagonist is an amount that is effective to treat a disease,disorder or unwanted physiological condition.

“Pain” is a sensory experience perceived by nerve tissue distinct fromsensations of touch, pressure, heat and cold. The range of painsensations, as well as the variation of perception of pain byindividuals, renders a precise definition of pain near impossible. Inthe context of the present invention, “pain” is used in the broadestpossible sense and includes nociceptive pain, such as pain related totissue damage and inflammation, pain related to noxious stimuli, acutepain, chronic pain, and neuropathic pain.

“Acute pain” is often short-lived with a specific cause and purpose;generally produces no persistent psychological reactions. Acute pain canoccur during soft tissue injury, and with infection and inflammation. Itcan be modulated and removed by treating its cause and through combinedstrategies using analgesics to treat the pain and antibiotics to treatthe infection.

“Chronic pain” is distinctly different from and more complex than acutepain. Chronic pain has no time limit, often has no apparent cause andserves no apparent biological purpose. Chronic pain can trigger multiplepsychological problems that confound both patient and health careprovider, leading to feelings of helplessness and hopelessness. The mostcommon causes of chronic pain include low-back pain, headache, recurrentfacial pain, pain associated with cancer and arthritis pain.

The pain is termed “neuropathic” when it is taken to representneurologic dysfunction. “Neuropathic pain” has a complex and variableetiology. It is typically characterized by hyperalgesia (lowered painthreshold and enhanced pain perception) and by allodynia (pain frominnocuous mechanical or thermal stimuli). Neuropathic pain is usuallychronic and tends not to respond to the same drugs as “normal pain”(nociceptive pain), therefore, its treatment is much more difficult.Neuropathic pain may develop whenever nerves are damaged, by trauma, bydisease such as diabetes, herpes zoster, or late-stage cancer, or bychemical injury (e.g., as an untoward consequence of agents includingthe false-nucleotide anti-HIV drugs). It may also develop afteramputation (including mastectomy). Examples of neuropathic pain includemonoradiculopathies, trigeminal neuralgia, postherpetic neuralgia,complex regional pain syndromes and the various peripheral neuropathies.This is in contrast with “normal pain” or “nociceptive pain,” whichincludes normal post-operative pain, pain associated with trauma, andchronic pain of arthritis.

“Peripheral neuropathy” is a neurodegenerative disorder that affects theperipheral nerves, most often manifested as one or a combination ofmotor, sensory, sensorimotor, or autonomic dysfunction. Peripheralneuropathies may, for example, be characterized by the degeneration ofperipheral sensory neurons, which may result from a disease or disordersuch as diabetes (diabetic neuropathy), alcoholism and acquiredimmunodeficiency syndrome (AIDS), from therapy such as cytostatic drugtherapy in cancer, or from genetic predisposition. Genetically acquiredperipheral neuropathies include, for example, Krabbe's disease,Metachromatic leukodystrophy, and Charcot-Marie-Tooth (CMT) Disease.Peripheral neuropathies are often accompanied by pain.

“Pharmaceutically acceptable” carriers, excipients, or stabilizers areones which are nontoxic to the cell or mammal being exposed thereto atthe dosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution such as phosphatebuffer or citrate buffer. The physiologically acceptable carrier mayalso comprise one or more of the following: antioxidants includingascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone, amino acids,carbohydrates including glucose, mannose, or dextrins, chelating agentssuch as EDTA, sugar alcohols such as mannitol or sorbitol, salt-formingcounterions such as sodium, and nonionic surfactants such as Tween™,polyethylene glycol (PEG), and Pluronics™.

“Peptide mimetics” are molecules which serve as substitutes for peptidesin interactions with the receptors of the present invention (Morgan etal., Ann. Reports Med. Chem. 24:243-252 (1989)). Peptide mimetics, asused herein, include synthetic structures that retain the structural andfunctional features of a peptide. Peptide mimetics may or may notcontain amino acids and/or peptide bonds. The term, “peptide mimetics”also includes peptoids and oligopeptoids, which are peptides oroligomers of N-substituted amino acids (Simon et al., Proc. Natl. Acad.Sci. USA 89:9367-9371 (1972)). Further included as peptide mimetics arepeptide libraries, which are collections of peptides designed to be of agiven amino acid length and representing all conceivable sequences ofamino acids corresponding thereto.

A. Proteins Expressed in Primary Sensory Neurons of Dorsal Root Ganglia

The present invention provides isolated mrg and drg-12 proteins, allelicvariants of the proteins, and conservative amino acid substitutions ofthe proteins. Polypeptide sequences of several Mrg proteins of thepresent invention are provided in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16,18, 21, 23, 25, 27, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107 and 109. Polypeptide sequences ofseveral drg-12 proteins of the present invention are provided in SEQ IDNOs: 14, 19 and 29.

The proteins of the present invention further include insertion,deletion or conservative amino acid substitution variants of thesequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107 and 109.

Ordinarily, the variants, allelic variants, the conservativesubstitution variants, and the members of the protein family, includingcorresponding homologues in other species, will have an amino acidsequence having at least about 50%, or about 60% to 75% amino acidsequence identity with the sequences set forth in SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109,more preferably at least about 80%, even more preferably at least about90%, and most preferably at least about 95% sequence identity with saidsequences.

The proteins of the present invention include molecules having the aminoacid sequence disclosed in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107 and 109; fragments thereof havinga consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30,35 or more amino acid residues of the protein; amino acid sequencevariants wherein one or more amino acid residues has been inserted N- orC-terminal to, or within, the disclosed coding sequence; and amino acidsequence variants of the disclosed sequence, or their fragments asdefined above, that have been substituted by another residue. Suchfragments, also referred to as peptides or polypeptides, may containantigenic regions, functional regions of the protein identified asregions of the amino acid sequence which correspond to known proteindomains, as well as regions of pronounced hydrophilicity. The regionsare all easily identifiable by using commonly available protein sequenceanalysis software such as MACVECTOR™ (Oxford Molecular).

Contemplated variants further include those containing predeterminedmutations by, e.g., homologous recombination, site-directed or PCRmutagenesis, and the corresponding proteins of other animal species,including but not limited to rabbit, rat, porcine, bovine, ovine,equine, human and non-human primate species, and the alleles or othernaturally occurring variants of the family of proteins; and derivativeswherein the protein has been covalently modified by substitution,chemical, enzymatic, or other appropriate means with a moiety other thana naturally occurring amino acid (for example a detectable moiety suchas an enzyme or radioisotope).

Protein domains such as a ligand binding domain, an extracellulardomain, a transmembrane domain (e.g. comprising seven membrane spanningsegments and cytosolic loops or two membrane spanning domains andcytosolic loops), the transmembrane domain and a cytoplasmic domain andan active site may all be found in the proteins or polypeptides of theinvention. Such domains are useful for making chimeric proteins and forin vitro assays of the invention.

Variations in native sequence proteins of the present invention or invarious domains identified therein, can be made, for example, using anytechniques known in the art. Variation can be achieved, for example, bysubstitution of at least one amino acid with any other amino acid in oneor more of the domains of the protein. A change in the amino acidsequence of a protein of the invention as compared with a nativesequence protein may be produced by a substitution, deletion orinsertion of one or more codons encoding the protein. A comparison ofthe sequence of the Mrg or drg-12 polypeptide to be changed with that ofhomologous known protein molecules may provide guidance as to whichamino acid residues may be inserted, substituted or deleted withoutaffecting a desired biological activity. In particular, it may bebeneficial to minimize the number of amino acid sequence changes made inregions of high homology. Amino acid substitutions can be the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

Polypeptide fragments are also useful in the methods of the presentinvention. Such fragments may be truncated at the N-terminus orC-terminus, or may lack internal residues, for example, when comparedwith a full-length native protein. Certain fragments lack amino acidresidues that are not essential for a desired biological activity of theMrg or drg-12 polypeptide.

Mrg or drg-12 fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized or generated by enzymatic digestion, such as by treating theprotein with an enzyme known to cleave proteins at sites defined byparticular amino acid residues. Alternatively, the DNA encoding theprotein may be digested with suitable restriction enzymes and thedesired fragment isolated. Yet another suitable technique involvesisolating and amplifying a DNA fragment encoding a desired polypeptidefragment, by polymerase chain reaction (PCR). Oligonucleotides thatdefine the desired termini of the DNA fragment are employed at the 5′and 3′ primers in the PCR. Preferably, Mrg or drg-12 polypeptidefragments share at least one biological and/or immunological activitywith a native Mrg or drg-12 polypeptide, respectively.

In making amino acid sequence variants that retain the requiredbiological properties of the corresponding native sequences, thehydropathic index of amino acids may be considered. For example, it isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score without significant changein biological activity. Thus, isoleucine, which has a hydropathic indexof +4.5, can generally be substituted for valine (+4.2) or leucine(+3.8), without significant impact on the biological activity of thepolypeptide in which the substitution is made. Similarly, usually lysine(−3.9) can be substituted for arginine (−4.5), without the expectationof any significant change in the biological properties of the underlyingpolypeptide. Other considerations for choosing amino acid substitutionsinclude the similarity of the side-chain substituents, for example,size, electrophilic character, charge in various amino acids. Ingeneral, alanine, glycine and serine; arginine and lysine; glutamate andaspartate; serine and threonine; and valine, leucine and isoleucine areinterchangeable, without the expectation of any significant change inbiological properties. Such substitutions are generally referred to asconservative amino acid substitutions, and are the preferred type ofsubstitutions within the polypeptides of the present invention.

Non-conservative substitutions will entail exchanging a member of oneclass of amino acids for another class. Such substituted residues alsomay be introduced into the conservative substitution sites or, morepreferably, into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such assite-directed mutagenesis, alanine scanning mutagenesis, and PCRmutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res.,13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)),cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restrictionselection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA,317:415 (1986)) or other known techniques can be performed on cloned DNAto produce the Mrg or drg-12 variant DNA.

Scanning amino acid analysis can be employed to identify one or moreamino acids that can be replaced without a significant impact onbiological activity. Among the preferred scanning amino acids arerelatively small, neutral amino acids. Such amino acids include alanine,glycine, serine, and cysteine. Alanine is preferred because, in additionto being the most common amino acid, it eliminates the side-chain beyondthe beta-carbon and is therefore less likely to alter the main-chainconformation of the variant (Cunningham and Wells, Science, 244:1081-1085 (1989)). Further, alanine is frequently found in both buriedand exposed positions (Creighton, The Proteins, (W. H. Freeman & Co.,N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitutiondoes not yield adequate amounts of variation, an isoteric amino acid canbe used.

As described below, members of the family of proteins can be used: 1) toidentify agents which modulate at least one activity of the protein; 2)to identify binding partners for the protein, 3) as an antigen to raisepolyclonal or monoclonal antibodies, 4) as a therapeutic target, 5) asdiagnostic markers to specific populations of pain sensing neurons and6) as targets for structure based ligand identification.

B. Nucleic Acid Molecules

The present invention further provides nucleic acid molecules thatencode the mrg or drg-12 proteins having SEQ ID NO: 2, 4, 6, 8, 10, 12,14, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109 and therelated polypeptides herein described, preferably in isolated form.cDNAs encoding eight full-length variants of Mrg receptors (mMrgA1-8)are provided in FIG. 6A (SEQ ID NO: 1, 3, 5, 11, 20, 22, 24, 26).

Preferred molecules are those that hybridize under the above definedstringent conditions to the complement of SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 20, 22, 24, 26 or 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 7274, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108 and whichencode a functional peptide. Preferred hybridizing molecules are thosethat hybridize under the above conditions to the complement strand ofthe open reading frame or coding sequences of SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 20, 22, 24, 26 or 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 7274, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108.

It is not intended that the methods of the present invention be limitedby the source of the polynucleotide. The polynucleotide can be from ahuman or non-human mammal, derived from any recombinant source,synthesized in vitro or by chemical synthesis. The nucleotide may be DNAor RNA and may exist in a double-stranded, single-stranded or partiallydouble-stranded form.

Nucleic acids useful in the present invention include, by way of exampleand not limitation, oligonucleotides such as antisense DNAs and/or RNAs;ribozymes; DNA for gene therapy; DNA and/or RNA chimeras; variousstructural forms of DNA including single-stranded DNA, double-strandedDNA, supercoiled DNA and/or triple-helix DNA; Z-DNA; and the like. Thenucleic acids may be prepared by any conventional means typically usedto prepare nucleic acids in large quantity. For example, DNAs and RNAsmay be chemically synthesized using commercially available reagents andsynthesizers by methods that are well-known in the art (see, e.g., Gait,1985, Oligonucleotide Synthesis: A Practical Approach, IRL Press,Oxford, England).

Any mRNA transcript encoded by Mrg or drg-12 nucleic acid sequences maybe used in the methods of the present invention, including inparticular, mRNA transcripts resulting from alternative splicing orprocessing of mRNA precursors.

Nucleic acids having modified nucleoside linkages may also be used inthe methods of the present invention. Modified nucleic acids may, forexample, have greater resistance to degradation. Such nucleic acids maybe synthesized using reagents and methods that are well known in theart. For example, methods for synthesizing nucleic acids containingphosphonate phosphorothioate, phosphorodithioate, phosphoramidatemethoxyethyl phosphoramidate, formacetal, thioformacetal,diisopropylsilyl, acetamidate, carbamate, dimethylene-sulfide(—CH₂—S—CH₂), dimethylene-sulfoxide (—CH₂—SO—CH₂), dimethylene-sulfone(—CH₂—SO₂—CH₂), 2′-O-alkyl, and 2′-deoxy-2′-fluoro phosphorothioateinternucleoside linkages are well known in the art.

In some embodiments of the present invention, the nucleotide used is anα-anomeric nucleotide. An α-anomeric nucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The nucleotide may be a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Means for purifying the nucleic acids of the present invention are wellknown in the art and the skilled artisan will be able to choose the mostappropriate method of purification for the particular circumstances.Such a choice may be made, in part, based on the size of the DNA, theamount to be purified and the desired purity. For example, the nucleicacids can be purified by reverse phase or ion exchange HPLC, sizeexclusion chromatography or gel electrophoresis.

Isolated or purified polynucleotides having at least 10 nucleotides(i.e., a hybridizable portion) of an Mrg or drg-12 coding sequence orits complement may also be used in the methods of the present invention.In other embodiments, the polynucleotides contain at least 25(continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150nucleotides, or 200 nucleotides of an Mrg coding sequence, or afull-length Mrg coding sequence. Nucleic acids can be single or doublestranded. Additionally, the invention relates to polynucleotides thatselectively hybridize to a complement of the foregoing coding sequences.In preferred embodiments, the polynucleotides contain at least 10, 25,50, 100, 150 or 200 nucleotides or the entire length of an Mrg codingsequence.

Nucleotide sequences that encode a mutant of an Mrg protein, peptidefragments of Mrg, truncated forms of Mrg, and Mrg fusion proteins mayalso be useful in the methods of the present invention. Nucleotidesencoding fusion proteins may include, but are not limited to, fulllength Mrg sequences, truncated forms of Mrg, or nucleotides encodingpeptide fragments of Mrg fused to an unrelated protein or peptide, suchas for example, a domain fused to an Ig Fc domain or fused to an enzymesuch as a fluorescent protein or a luminescent protein which can be usedas a marker.

Furthermore, polynucleotide variants that have been generated, at leastin part, by some form of directed evolution, such as gene shuffling orrecursive sequence recombination may be used in the methods of thepresent invention. For example, using such techniques novel sequencescan be generated encoding proteins similar to Mrg or drg-12 but havingaltered functional or structural characteristics.

Highly related gene homologs of the Mrg encoding polynucleotidesequences described above may also be useful in the present invention.Highly related homologs can encode proteins sharing functionalactivities with Mrg proteins.

The present invention further provides fragments of the encoding nucleicacid molecule. Fragments of the encoding nucleic acid molecules of thepresent invention (i.e., synthetic oligonucleotides) that are used asprobes or specific primers for the polymerase chain reaction (PCR), orto synthesize gene sequences encoding proteins of the invention, caneasily be synthesized by chemical techniques, for example, thephosphotriester method of Matteucci, et al., (J. Am. Chem. Soc.103:3185-3191, 1981) or using automated synthesis methods. In addition,larger DNA segments can readily be prepared by well known methods, suchas synthesis of a group of oligonucleotides that define various modularsegments of the gene, followed by ligation of oligonucleotides to buildthe complete modified gene.

The encoding nucleic acid molecules of the present invention may furtherbe modified so as to contain a detectable label for diagnostic and probepurposes. A variety of such labels are known in the art and can readilybe employed with the encoding molecules herein described. Suitablelabels include, but are not limited to, biotin, radiolabeled nucleotidesand the like. A skilled artisan can readily employ any such label toobtain labeled variants of the nucleic acid molecules of the invention.

Any nucleotide sequence which encodes the amino acid sequence of aprotein of the invention can be used to generate recombinant moleculeswhich direct the expression of the protein, as described in more detailbelow. In addition, the methods of the present invention may alsoutilize a fusion polynucleotide comprising an Mrg or drg-12 codingsequence and a second coding sequence for a heterologous protein.

C. Isolation of Other Related Nucleic Acid Molecules

As described above, the identification and characterization of a nucleicacid molecule encoding an mrg or drg-12 protein allows a skilled artisanto isolate nucleic acid molecules that encode other members of the sameprotein family in addition to the sequences herein described

Essentially, a skilled artisan can readily use the amino acid sequenceof SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107 or 109 to generate antibody probes to screen expressionlibraries prepared from appropriate cells. Typically, polyclonalantiserum from mammals such as rabbits immunized with the purifiedprotein (as described below) or monoclonal antibodies can be used toprobe a mammalian cDNA or genomic expression library, such as a lambdagtl1 library, to obtain the appropriate coding sequence for othermembers of the protein family. The cloned cDNA sequence can be expressedas a fusion protein, expressed directly using its own control sequences,or expressed by constructions using control sequences appropriate to theparticular host used for expression of the protein.

Alternatively, a portion of the coding sequence herein described can besynthesized and used as a probe to retrieve DNA encoding a member of theMrg protein family from cells derived from any mammalian organism,particularly cells believed to express Mrg proteins. Oligomerscontaining approximately 18-20 nucleotides (encoding about a 6-7 aminoacid stretch) are prepared and used to screen genomic DNA or cDNAlibraries to obtain hybridization under stringent conditions orconditions of sufficient stringency to eliminate an undue level of falsepositives. Oligonucleotides corresponding to either the 5′ or 3′terminus of the coding sequence may be used to obtain longer nucleotidesequences.

It may be necessary to screen multiple cDNA libraries to obtain afull-length cDNA. In addition, it may be necessary to use a techniquesuch as the RACE (Rapid Amplification of cDNA Ends) technique to obtainthe complete 5′ terminal coding region. RACE is a PCR-based strategy foramplifying the 5′ end of incomplete cDNAs. To obtain the 5′ end of thecDNA, PCR is carried out on 5′-RACE-Ready cDNA using an anchor primerand a 3′ primer. A second PCR is then carried out using the anchoredprimer and a nested 3′ primer. Once a full length cDNA sequence isobtained, it may be translated into amino acid sequence and examined foridentifiable regions such as a continuous open reading frame flanked bytranslation initiation and termination sites, a potential signalsequence and finally overall structural similarity to the proteinsequences disclosed herein.

Related nucleic acid molecules may also be retrieved by using pairs ofoligonucleotide primers in a polymerase chain reaction (PCR) toselectively clone an encoding nucleic acid molecule. The oligonucleotideprimers may be degenerate oligonucleotide primer pools designed on thebasis of the protein coding sequences disclosed herein. The template forthe reaction may be cDNA obtained by reverse transcription (RT) of mRNAprepared from, for example, human or non-human cell lines or tissuesknown or suspected to express an Mrg or drg-12 gene allele, such as DRGtissue. A PCR denature/anneal/extend cycle for using such PCR primers iswell known in the art and can readily be adapted for use in isolatingother encoding nucleic acid molecules.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an Mrg or drg-12 codingsequence. The PCR fragment may then be used to isolate a full-lengthcDNA clone by a variety of methods. For example, the amplified fragmentmay be labeled and used to screen a cDNA library. Alternatively, thelabeled fragment may be used to isolate genomic clones via the screeningof a genomic library.

PCR technology may also be utilized to isolate full-length cDNAsequences. RNA may be isolated, from an appropriate cellular or tissuesource, such as dorsal root ganglion (DRG) and an RT reaction may becarried out using an oligonucleotide primer specific for the most 5′ endof the amplified fragment to prime first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” with guanines in a terminaltransferase reaction, the hybrid may be digested with RNAase H, andsecond strand synthesis may then be primed with a poly-C primer. Thisallows isolation of cDNA sequences upstream of the amplified fragment.

Nucleic acid molecules encoding other members of the mrg and drg-12families may also be identified in existing genomic or other sequenceinformation using any available computational method, including but notlimited to: PSI-BLAST (Altschul, et al. (1997) Nucleic Acids Res.25:3389-3402); PHI-BLAST (Zhang, et al. (1998), Nucleic Acids Res.26:3986-3990), 3D-PSSM (Kelly et al. J. Mol. Biol. 299(2): 499-520(2000)); and other computational analysis methods (Shi et al. Biochem.Biophys. Res. Commun. 262(1):132-8 (1999) and Matsunami et. al. Nature404(6778):601-4 (2000).

A cDNA clone of a mutant or allelic variant of an Mrg or drg-12 gene mayalso be isolated. A possible source of a mutant or variant protein istissue known to express Mrg or drg-12, such as DRG tissue, obtained froman individual putatively carrying a mutant or variant form of Mrg ordrg-12. Such an individual may be identified, for example, by ademonstration of increased or decreased responsiveness to painfulstimuli. In one embodiment, a mutant or variant Mrg or drg-12 gene maybe identified by PCR. The first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to mRNA isolated from the tissueputatively carrying a variant and extending the new strand with reversetranscriptase. The second strand of the cDNA is then synthesized usingan oligonucleotide that hybridizes specifically to the 5′ end of thenormal gene. Using these two primers, the product is then amplified viaPCR, cloned into a suitable vector, and subjected to DNA sequenceanalysis through methods well known to those of skill in the art. Bycomparing the DNA sequence of the mutant Mrg allele to that of thenormal Mrg allele, the mutation(s) responsible for any loss oralteration of function of the mutant Mrg gene product can beascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry a mutant Mrg allele,or a cDNA library can be constructed using RNA from a tissue known, orsuspected, to express a mutant Mrg allele. An unimpaired Mrg gene or anysuitable fragment thereof may then be labeled and used as a probe toidentify the corresponding mutant Mrg allele in such libraries. Clonescontaining the mutant Mrg gene sequences may then be purified andsubjected to sequence analysis according to methods well known to thoseof skill in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant Mrg allele in an individual suspected ofcarrying such a mutant allele. In this manner, gene products made by theputatively mutant tissue may be expressed and screened using standardantibody screening techniques in conjunction with antibodies raisedagainst the normal Mrg gene product, as described, below.

D. Recombinant DNA Molecules Containing a Nucleic Acid Molecule

The present invention further provides recombinant DNA molecules (rDNAs)that contain a coding sequence. As used herein, a rDNA molecule is a DNAmolecule that has been subjected to molecular manipulation in situ.Methods for generating rDNA molecules are well known in the art, forexample, see Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd edition, 1989; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. In the preferred rDNA molecules, a coding DNA sequence isoperably linked to expression control sequences and/or vector sequences.

Thus the present invention also contemplates DNA vectors that containany of the Mrg or drg-12 coding sequences and/or their complements,optionally associated with a regulatory element that directs theexpression of the coding sequences. The choice of vector and/orexpression control sequences to which one of the protein family encodingsequences of the present invention is operably linked depends directly,as is well known in the art, on the functional properties desired, e.g.,protein expression, and the host cell to be transformed. A vectorcontemplated by the present invention is at least capable of directingthe replication or insertion into the host chromosome, and preferablyalso expression, of the structural gene included in the rDNA molecule.

Both cloning and expression vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Incloning vectors this sequence is one that enables the vector toreplicate independently of the host chromosomal DNA, and includesorigins of replication or autonomously replicating sequences. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

In addition to being capable of replication in at least one class oforganism most expression vectors can be transfected into anotherorganism for expression. For example, a vector is replicated in E. coliand then the same vector is transfected into yeast or mammalian cellsfor expression.

DNA may also be amplified by insertion into the host genome. Forexample, transfection of Bacillus with a vector comprising a DNAsequence complementary to a Bacillus genomic sequence results inhomologous recombination with the genome and insertion of the DNA fromthe vector. One disadvantage to this type of system is that the recoveryof genomic DNA encoding the protein of interest is more complex thanthat of an exogenously replicated vector because restriction enzymedigestion is required to excise the DNA.

Expression control elements that are used for regulating the expressionof an operably linked protein encoding sequence are known in the art andinclude, but are not limited to, inducible promoters, constitutivepromoters, secretion signals, and other regulatory elements. Preferably,the inducible promoter is readily controlled, such as being responsiveto a nutrient in the host cell's medium.

In one embodiment, the vector containing a coding nucleic acid moleculewill include a prokaryotic replicon, i.e., a DNA sequence having theability to direct autonomous replication and maintenance of therecombinant DNA molecule extrachromosomally in a prokaryotic host cell,such as a bacterial host cell, transformed therewith. Such replicons arewell known in the art. In addition, vectors that include a prokaryoticreplicon may also include a gene whose expression confers a detectablemarker such as a drug resistance. Typical bacterial drug resistancegenes are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can further include aprokaryotic or bacteriophage promoter capable of directing theexpression (transcription and translation) of the coding gene sequencesin a bacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences that arecompatible with bacterial hosts are typically provided in plasmidvectors containing convenient restriction sites for insertion of a DNAsegment of the present invention. Typical of such vector plasmids arepUC8, pUC9, pBR322 and pBR329 available from BioRad Laboratories,(Richmond, Calif.), pPL and pKK223 available from Pharmacia (Piscataway,N.J.).

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form rDNAmolecules that contain a coding sequence. Eukaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. Typically, such vectors are provided containingconvenient restriction sites for insertion of the desired DNA segment.Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d(International Biotechnologies, Inc.), pTDT1 (ATCC, #31255), eukaryoticviral vectors such as adenoviral or retroviral vectors, and the likeeukaryotic expression vectors.

Eukaryotic cell expression vectors used to construct the rDNA moleculesof the present invention may further include a selectable marker that iseffective in an eukaryotic cell, preferably a drug resistance selectionmarker. This gene encodes a factor necessary for the survival or growthof transformed host cells grown in a selective culture medium. Hostcells not transformed with the vector containing the selection gene willnot survive in the culture medium. Typical selection genes encodeproteins that confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, complementauxotrophic deficiencies, or supply critical nutrients withheld from themedia. A preferred drug resistance marker is the gene whose expressionresults in neomycin resistance, i.e., the neomycin phosphotransferase(neo) gene. (Southern et al., J. Mol. Anal. Genet. 1:327-341, 1982.) Theselectable marker can optionally be present on a separate plasmid andintroduced by co-transfection.

In one example of a selection system, mammalian cell transformants areplaced under selection pressure such that only the transformants areable to survive by virtue of having taken up the vector(s). Selectionpressure is imposed by progressively increasing the concentration ofselection agent in the culture medium, thereby stimulating amplificationof both the selection gene and the DNA that encodes the desired protein.Amplification is the process by which genes in greater demand for theproduction of a protein critical for growth are reiterated in tandemwithin the chromosomes of successive generations of recombinant cells.Increased quantities of the desired protein, such as Mrg, aresynthesized from the amplified DNA. Examples of amplifiable genesinclude DHFR, thymidine kinase, metallothionein-I and -II, adenosinedeaminase, and ornithine decarboxylase.

Thus in one embodiment Chinese hamster ovary (CHO) cells deficient inDHFR activity are prepared and propagated as described by Urlaub et al.,Proc. Natl. Acad. Sci. USA, 77:4216 (1980). The CHO cells are thentransformed with the DHFR selection gene and transformants are areidentified by culturing in a culture medium that contains methotrexate(Mtx), a competitive antagonist of DHFR. The transformed cells are thenexposed to increased levels of methotrexate. This leads to the synthesisof multiple copies of the DHFR gene, and, concomitantly, multiple copiesof other DNA comprising the expression vectors, such as the DNA encodingthe protein of interest, for example DNA encoding Mrg.

Alternatively, host cells can be transformed or co-transformed with DNAsequences encoding a protein of interest such as Mrg, wild-type DHFRprotein, and another selectable marker such as aminoglycoside3′-phosphotransferase (APH). The transformants can then be selected bygrowth in medium containing a selection agent for the selectable markersuch as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, orG418.

As mentioned above, expression and cloning vectors usually contain apromoter that is recognized by the host organism and is operably linkedto the nucleic acid encoding the protein of interest. Promoters areuntranslated sequences located upstream (5′) to the start codon of astructural gene (generally within about 100 to 1000 bp) and control thetranscription and translation of the particular nucleic acid sequence,such as an Mrg nucleic acid sequence, to which they are operably linked.Promoters may be inducible or constitutive. Inducible promoters initiateincreased levels of transcription from DNA under their control inresponse to some change in culture conditions, such as a change intemperature. Many different promoters are well known in the art, as aremethods for operably linking the promoter to the DNA encoding theprotein of interest. Both the native Mrg or drg-12 promoter sequence andmany heterologous promoters may be used to direct amplification and/orexpression of the Mrg or drg-12 DNA. However, heterologous promoters arepreferred, as they generally permit greater transcription and higheryields of the desired protein as compared to the native promoter.

Promoters suitable for use with prokaryotic hosts include, for example,the β-lactamase and lactose promoter systems (Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)). However, otherbacterial promoters are well known in the art and are suitable.Promoters for use in bacterial systems also will contain aShine-Delgarno (S.D.) sequence operably linked to the DNA encoding theprotein of interest.

Promoter sequences that can be used in eukaryotic cells are also wellknown. Virtually all eukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the transcription initiationsite. Another sequence found 70 to 80 bases upstream from the start oftranscription of many genes is a CXCAAT region where X may be anynucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequencethat may be the signal for addition of the poly-A tail to the 3′ end ofthe coding sequence. All of these sequences may be inserted intoeukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)).

Inducible promoters for use with yeast are also well known and includethe promoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in EP73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Mrg or drg-12 transcription from vectors in mammalian host cells mayalso be controlled by promoters obtained from the genomes of virusessuch as polyoma virus, fowlpox virus, adenovirus, bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and most preferably Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, and from the promoter normallyassociated with the native sequence, provided such promoters arecompatible with the host cell systems.

Transcription may be increased by inserting an enhancer sequence intothe vector. Enhancers are cis-acting elements of DNA, usually about 10to 300 bp in length, that act on a promoter to increase itstranscription. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, α-fetoprotein, and insulin).Preferably an enhancer from a eukaryotic cell virus will be used.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the protein-encoding sequence, but is preferablylocated at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. These sequences are oftenfound in the 5′ and, occasionally 3′, untranslated regions of eukaryoticor viral DNAs or cDNAs and are well known in the art.

Plasmid vectors containing one or more of the components described aboveare readily constructed using standard techniques well known in the art.

For analysis to confirm correct sequences in plasmids constructed, theplasmid may be replicated in E. coli, purified, and analyzed byrestriction endonuclease digestion, and/or sequenced by conventionalmethods.

Particularly useful in the preparation of proteins of the presentinvention are expression vectors that provide for transient expressionin mammalian cells of DNA encoding Mrg or drg-12. Transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa the polypeptide encoded by the expression vector. Sambrook et al.,supra, pp. 16.17-16.22. Transient expression systems allow for theconvenient positive identification of polypeptides encoded by clonedDNAs, as well as for the screening of such polypeptides for desiredbiological or physiological properties. Thus, transient expressionsystems are particularly useful in the invention for purposes ofidentifying biologically active analogs and variants of the polypeptidesof the invention and for identifying agonists and antagonists thereof.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of Mrg or drg-12 in recombinant vertebrate cell culture arewell known in the art and are readily adapted to the specificcircumstances.

E. Host Cells Containing an Exogenously Supplied Coding Nucleic AcidMolecule

The present invention further provides host cells transformed with anucleic acid molecule that encodes a protein of the present invention.The host cell can be either prokaryotic or eukaryotic but is preferablyeukaryotic.

Eukaryotic cells useful for expression of a protein of the invention arenot limited, so long as the cell line is compatible with cell culturemethods and compatible with the propagation of the expression vector andexpression of the gene product. Such host cells are capable of complexprocessing and glycosylation activities. In principle, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Preferred eukaryotic host cells include, but arenot limited to, yeast, insect and mammalian cells, preferably vertebratecells such as those from a mouse, rat, monkey or human cell line.Preferred eukaryotic host cells include Chinese hamster ovary (CHO)cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells(NIH/3T3) available from the ATCC as CRL 1658, baby hamster kidney cells(BHK), HEK293 cells and the like eukaryotic tissue culture cell lines.

Propagation of vertebrate cells in culture is a routine procedure. See,e.g., Tissue Culture, Academic Press, Kruse and Patterson, editors(1973). Additional examples of useful mammalian host cell lines that canbe readily cultured are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammarytumor (MMT 060562, ATCC CCL51).

Xenopus oocytes may also be directly injected with RNA capable ofexpressing either the mrg or drg-12 proteins by standard procedures (seeTominaga et al. Jpn J. Pharmacol. 83(1):20-4 (2000); Tominaga et al.Neuron 21(3):531-43 (1998) and Bisogno et al. Biochem, Biophys. Res.Commun. 262(1):275-84 (1999)).

Examples of invertebrate cells that can be used as hosts include plantand insect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells are known in the art and maybe utilized in the methods of the present invention. In addition, plantcell cultures are known and may be transfected, for example, byincubation with Agrobacterium tumefaciens, which has been manipulated tocontain Mrg or drg-12 encoding DNA.

Any prokaryotic host can be used to express a rDNA molecule encoding aprotein or a protein fragment of the invention. Suitable prokaryotesinclude eubacteria, such as Gram-negative or Gram-positive organisms,for example, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. The preferredprokaryotic host is E. coli. In addition, it is preferably that the hostcell secrete minimal amounts of proteolytic enzymes.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for Mrg- ordrg-12-encoding vectors. For example, Saccharomyces cerevisiae may beused. In addition a number of other genera, species, and strains arecommonly available and useful herein, such as Schizosaccharomyces pombe(Beach et al. Nature, 290:140 (1981); EP 139,383); Kluyveromyces hosts(U.S. Pat. No. 4,943,529; Fleer et al., supra) such as, e.g., K. lactis(MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737(1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., supra), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070;Sreekrishna et al. J. Basic Microbiol., 28:265-278 (1988)); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al. Proc.Natl. Acad. Sci. USA, 76:5259-5263 (1979)); Schwanniomyces such asSchwanniomyces occidentalis (EP 394,538); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357), andAspergillus hosts such as A. nidulans (Ballance et al. Biochem. Biophys.Res. Commun., 112:284-289 (1983); Tilburn et al., Gene, 26:205-221(1983); Yelton et al. Proc. Natl. Acad. Sci. USA, 81:1470-1474 (1984))and A. niger (Kelly et al. EMBO J., 4:475-479 (1985)).

Transformation of appropriate cell hosts with a rDNA molecule of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used and host system employed. With regardto transformation of prokaryotic host cells, electroporation and salttreatment methods are typically employed, see, for example, Cohen et al.Proc. Natl. Acad. Sci. USA 69:2110, (1972); and Maniatis et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982). With regard to transformation ofvertebrate cells with vectors containing rDNAs, electroporation,cationic lipid or salt treatment methods are typically employed, see,for example, Graham et al. Virol. 52:456, (1973); Wigler et al. Proc.Natl. Acad. Sci. USA 76:1373-76, (1979). The calcium phosphateprecipitation method is preferred. However, other methods of forintroducing DNA into cells may also be used, including nuclearmicroinjection and bacterial protoplast fusion.

For transient expression of recombinant channels, transformed host cellsfor the measurement of Na⁺ current or intracellular Na⁺ levels aretypically prepared by co-transfecting constructs into cells such asHEK293 cells. with a fluorescent reporter plasmid (such as pGreenLantern-1, Life Technologies) using the calcium-phosphate precipitationtechnique (Ukomadu et al. Neuron 8, 663-676 (1992)). After forty-eighthours, cells with green fluorescence are selected for recording(Dib-Hajj et al. FEBS Lett. 416, 11-14 (1997)). Similarly, for transientexpression of Mrg receptors and measurement of intracellular Ca²⁺changes in response to receptor activation as described in Example 4,HEK cells can be co-transfected with Mrg expression constructs and afluorescent reporter plasmid. HEK293 cells are typically grown in highglucose DMEM (Life Technologies) supplemented with 10% fetal calf serum(Life Technologies).

Prokaryotic cells used to produce polypeptides of this invention arecultured in suitable media as described generally in Sambrook et al.,supra.

The mammalian host cells used to produce the polypeptides of thisinvention may be cultured in a variety of media, including but notlimited to commercially available media such as Ham's F10 (Sigma),Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium ((DMEM), Sigma). In addition, any ofthe media described in Ham et al. Meth. Enz., 58:44 (1979), Barnes etal. Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S.Pat. Re. 30,985 may be used as culture media for the host cells. Any ofthese media may be supplemented as necessary with hormones and/or othergrowth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics, trace elements, and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations as determined by the skilled practitioner.The culture conditions are those previously used with the host cellselected for expression, and will be apparent to the skilled artisan.

The host cells referred to in this disclosure encompass cells in cultureas well as cells that are within a host animal.

Successfully transformed cells, i.e., cells that contain a rDNA moleculeof the present invention, can be identified by well known techniquesincluding the selection for a selectable marker. For example, cellsresulting from the introduction of an rDNA of the present invention canbe cloned to produce single colonies. Cells from those colonies can beharvested, lysed and their DNA content examined for the presence of therDNA using a method such as that described by Southern, J. Mol. Biol.98:503, (1975), or Berent et al., Biotech. 3:208, (1985) or the proteinsproduced from the cell assayed via an immunological method as describedbelow.

Gene amplification and/or expression may be measured by any techniqueknown in the art, including Southern blotting, Northern blotting toquantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci.USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. Immunological methods for measuringgene expression include immunohistochemical staining of tissue sectionsor cells in culture, as well as assaying protein levels in culturemedium or body fluids. With immunohistochemical staining techniques, acell sample is prepared by dehydration and fixation, followed byreaction with labeled antibodies specific for the gene product, wherethe labels are usually visually detectable, such as enzymatic labels,fluorescent labels, luminescent labels, and the like.

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared as described herein.

F. Production of Recombinant Proteins Using an rDNA Molecule

The present invention further provides methods for producing a proteinof the invention using nucleic acid molecules herein described. Ingeneral terms, the production of a recombinant form of a proteintypically involves the following steps:

A nucleic acid molecule is first obtained that encodes a mrg or drg-12protein of the invention, for example, nucleotides 115-1026 of SEQ IDNO: 1, nucleotides 115-1029 of SEQ ID NO: 1, nucleotides 137-1051 of SEQID NO: 3, nucleotides 137-1054 of SEQ ID NO: 3, nucleotides 165-1070 ofSEQ ID NO: 5, nucleotides 165-1073 of SEQ ID NO: 5, nucleotides 1-450 ofSEQ ID NO: 7, nucleotides 1-459 of SEQ ID NO: 9, nucleotides 1820-2734of SEQ ID NO: 11, nucleotides 170-574 of SEQ ID NO: 13, nucleotides170-577 of SEQ ID NO: 13, nucleotides 328-1293 of SEQ ID NO: 15,nucleotides 328-1296 of SEQ ID NO:15, nucleotides 171-1160 of SEQ ID NO:17, nucleotides 171-1163 of SEQ ID NO:17, nucleotides 83-943 of SEQ IDNO: 20, nucleotides 83-946 of SEQ ID NO:20; nucleotides 16-918 of SEQ IDNO: 22, nucleotides 16-921 of SEQ ID NO: 22; nucleotides 106-1020 of SEQID NO: 24, nucleotides 106-1023 of SEQ ID NO: 24; nucleotides 45-959 ofSEQ ID NO: 26, nucleotides 45-962 of SEQ ID NO: 26, nucleotides 1-405 ofSEQ ID NO: 28 and nucleotides 1-408 of SEQ ID NO: 28. If the encodingsequence is uninterrupted by introns, as are these sequences, it isdirectly suitable for expression in any host.

The nucleic acid molecule is then preferably placed in operable linkagewith suitable control sequences, as described above, to form anexpression unit containing the protein open reading frame. Theexpression unit is used to transform a suitable host and the transformedhost is cultured under conditions that allow the production of therecombinant protein. Optionally the recombinant protein is isolated fromthe medium or from the cells; recovery and purification of the proteinmay not be necessary in some instances where some impurities may betolerated or when the recombinant cells are used, for instance, in highthroughput assays.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using appropriate replicons and control sequences, as setforth above. The control sequences, expression vectors, andtransformation methods are dependent on the type of host cell used toexpress the gene and were discussed in detail earlier. Suitablerestriction sites can, if not normally available, be added to the endsof the coding sequence so as to provide an excisable gene to insert intothese vectors. A skilled artisan can readily adapt any host/expressionsystem known in the art for use with the nucleic acid molecules of theinvention to produce recombinant protein.

In one embodiment, Mrg or drg-12 may be produced by homologousrecombination. Briefly, primary human cells containing an Mrg- ordrg-12-encoding gene are transformed with a vector comprising anamplifiable gene (such as dihydrofolate reductase (DHFR)) and at leastone flanking region of a length of at least about 150 bp that ishomologous with a DNA sequence at the locus of the coding region of theMrg or drg-12 gene. The amplifiable gene must be located such that itdoes not interfere with expression of the Mrg or drg-12 gene. Upontransformation the construct becomes homologously integrated into thegenome of the primary cells to define an amplifiable region.

Transformed cells are then selected for by means of the amplifiable geneor another marker present in the construct. The presence of the markergene establishes the presence and integration of the construct into thehost genome. PCR, followed by sequencing or restriction fragmentanalysis. may be used to confirm that homologous recombination occurred.

The entire amplifiable region is then isolated from the identifiedprimary cells and transformed into host cells. Clones are then selectedthat contain the amplifiable region, which is then amplified bytreatment with an amplifying agent. Finally, the host cells are grown soas to express the gene and produce the desired protein.

The proteins of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide. In one embodiment the heterologous polypeptide may be asignal sequence. In general, the signal sequence may be a component ofthe vector, or it may be a part of the Mrg or drg-12 DNA that isinserted into the vector. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For expression in prokaryotic hostcells the signal sequence may be a prokaryotic signal sequence selected,for example, from the group consisting of the alkaline phosphatase,penicillinase, lpp, and heat-stable enterotoxin II leaders. For yeastsecretion the native signal sequence may be substituted by, e.g., theyeast invertase leader, α factor leader (including Saccharomyces andKluyveromyces α-factor leaders, or acid phosphatase leader and the C.albicans glucoamylase leader). In mammalian cell expression any nativesignal sequence is satisfactory. Alternatively it may be substitutedwith a signal sequence from related proteins, as well as viral secretoryleaders, for example, the herpes simplex gD signal. The DNA for suchprecursor regions is ligated in reading frame to DNA encoding the matureprotein or a soluble variant thereof.

The heterologous polypeptide may also be a marker polypeptide that canbe used, for example, to identify the location of expression of thefusion protein. The marker polypeptide may be any known in the art, suchas a fluorescent protein. A prefered marker protein is green fluorescentprotein (GFP).

G. Modifications of Mrg polypeptides

Covalent modifications of Mrg and drg-12 and their respective variantsare included within the scope of this invention. In one embodiment,specific amino acid residues of a polypeptide of the invention arereacted with an organic derivatizing agent. Derivatization withbifunctional agents is useful, for instance, for crosslinking Mrg or Mrgfragments or derivatives to a water-insoluble support matrix or surfacefor use in methods for purifying anti-Mrg antibodies and identifyingbinding partners and ligands. In addition, Mrg or Mrg fragments may becrosslinked to each other to modulate binding specificity and effectorfunction. Many crosslinking agents are known in the art and include, butare not limited to, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides such asbis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other contemplated modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Modification of the glycosylation patterns of the polypeptides of theinvention are also contemplated. Methods for altering the glycosylationpattern of polypeptides are well known in the art. For example, one ormore of the carbohydrate moities found in native sequence Mrg or drg-12may be removed chemically, enzymatically or by modifying theglycosylation site. Alternatively, additional gycosylation can be added,such as by manipulating the composition of the carbohydrate moitiesdirectly or by adding glycosylation sites not present in the nativesequence Mrg or drg-12 by altering the amino acid sequence.

Another type of covalent modification of the polypeptides of theinvention comprises linking the polypeptide or a fragment or derivativethereof to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The polypeptides of the present invention may also be modified in a wayto form a chimeric molecule comprising Mrg or drg-12 fused to another,heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theMrg or drg-12 with a tag polypeptide that provides an epitope to whichan anti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the polypeptide. Theepitope tag allows for identification of the chimeric protein as well aspurification of the chimeric protein by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. A number of tag polypeptides and their respectiveantibodies are well known in the art. Well known tags includepoly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags;the flue HA tag polypeptide (Field et al., Mol. Cell. Biol., 8:2159-2165(1988)); the c-myc tag (Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tag(Paborsky et al., Protein Engineering, 3(6):547-553 (1990)) and theFlag-peptide (Hopp et al., BioTechnology, 6:1204-1210 (1988)).

In another embodiment, the chimeric molecule comprises a fusion of Mrgor drg-12 with an immunoglobulin or a particular region of animmunoglobulin. To produce an immunoadhesin, the polypeptide of theinvention or a fragment or specific domain(s) thereof could be fused tothe Fc region of an IgG molecule. Typically the fusion is to animmunoglobulin heavy chain constant region sequence. Mrg- ordrg-12-immunoglobulin chimeras for use in the present invention arenormally prepared from nucleic acid encoding one or more extracellulardomains, or fragments thereof, of an Mrg or drg-12 receptor fusedC-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence. N-terminal fusions are alsopossible.

While not required in the immunoadhesins of the present invention, animmunoglobulin light chain might be present either covalently linked toan Mrg- or drg-12-immunoglobulin heavy chain fusion polypeptide, ordirectly fused to Mrg or drg-12. In order to obtain covalentassociation, DNA encoding an immunoglobulin light chain may becoexpressed with the DNA encoding the Mrg- or drg-12-immunoglobulinheavy chain fusion protein. Upon secretion, the hybrid heavy chain andthe light chain will be covalently associated to provide animmunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs.

Bispecific immunoadhesins may also be made. Such immunoadhesins maycombine an Mrg or drg-12 domain and a domain, such as the extracellulardomain, from another receptor. Alternatively, the immunoadhesins hereinmight comprise portions of two different Mrg receptors, each fused to animmunoglobulin heavy chain constant domain sequence.

In yet another embodiment, the chimeric molecule of the presentinvention comprises a fusion of Mrg or drg-12 or a fragment or domain(s)thereof, with a heterologous receptor or fragment or domain(s) thereof.The heterologous receptor may be a related Mrg or drg-12 family member,or may be completely unrelated. The heterologous protein fused to theMrg or drg-12 protein may be chosen to obtain a fusion protein with adesired ligand specificity or a desired affinity for a particular ligandor to obtain a fusion protein with a desired effector function.

H. Methods of Using mrgs or drgs as Molecular or Diagnostic Probes

The sequences and antibodies, proteins and peptides of the presentinvention may be used as molecular probes for the detection of cells ortissues related to or involved with sensory perception, especiallyperception of pain. Although many methods may be used to detect thenucleic acids or proteins of the invention in situ, preferred probesinclude antisense molecules and anti-mrg or anti-drg-12 antibodies.

Probes for the detection of the nucleic acids or proteins of theinvention may find use in the identification of the involvement of Mrgor drg-12 proteins in particular disease states, such as glaucoma orchronic pain, or in enhanced or inhibited sensory perception. Inparticular, probes of the present invention may be useful in determiningif Mrg or drg-12 expression is increased or decreased in patientsdemonstrating changes in sensory perception, such as in patients withallodynia, hyperalgesia or chronic pain, or patients with a disease ordisorder, such as glaucoma. A determination of decreased expression oroverexpression of a polypeptide of the invention may be useful inidentifying a therapeutic approach to treating the disorder, such as byadministering Mrg or drg-12 agonists or antagonists.

Determination of changes in Mrg or drg-12 expression levels in animalmodels of disease states, particularly pain, may also be useful inidentifying the types of disorders that might be effectively treated bycompounds that modify expression or activity.

Further, the probes of the invention, including antisense molecules andantibodies, may be used to detect the expression of mutant or variantforms of Mrg or drg-12 variants. The ability to detect such variants maybe useful in identifying the role that the variants play in particulardisease states and in the symptoms experienced by particular patients.Identification of the involvement of a variant of Mrg or drg-12 in adisease or disorder may suggest a therapeutic approach for treatment ofthe disease or disorder, such as gene therapy or the administration ofagonists or antagonists known to bind the receptor variant.

In addition, probes of the invention may be used to determine the exactexpression patterns of the various Mrg and drg-12 family members,including the relationship of one to another. For example, themicroscopy images of in situ hybridization in FIG. 2 show thelocalization of antisense staining against a nucleotide of SEQ ID NO:2(“mrg3”) and of SEQ ID NO:4 (“mrg4”) in transverse sections of dorsalroot ganglia (DRG) from newborn wild type (WT) and Neurogenin1 nullmutant (Ngn1^(−/−)) mice. White dashed lines outline the DRG and blackdashed lines outline the spinal cord. Note that in the Ngn1^(−/−)mutant, the size of the DRG is severely reduced due to the loss ofnociceptive sensory neurons, identified using three other independentmarkers (trkA; VR-1 and SNS-TTXi (Ma et al., (1999)). mrg3 is expressedin a subset of DRG in WT mice (A) but is absent in the Ngn1^(−/−) DRG(B). mrg4 is expressed in a smaller subset of DRG than that of mrg3 (C).It is also absent in the Ngn1^(−/−) DRG (D). The loss of mrg-expressingneurons in the Ngn1^(−/−) DRG indicates that these neurons are likely tobe nociceptive.

Expression of mrgs in subsets of dorsal root ganglia (DRG) neurons areshown in FIG. 2A. Frozen transverse sections of DRG from wild-type (a-i)and ngn1^(−/−) (j) mutant new born mice were annealed with antisensedigoxigenin RNA probes, and hybridization was visualized with analkaline phosphatase-conjugated antibody. Positive signals are shown asdark purple stainings. TrkA is expressed in a large portion of wild-typeDRG neurons (a) but absent in ngn1^(−/−) (data not shown). Each of theeight mrg genes (b-i) is expressed in a small subset of neurons inwild-type DRG in completely absent in ngn1^(−/−) DRG (j and data notshown). Black dash line outlines the ngn1^(−/−) mutant DRG.

In FIG. 2B, mrgs are expressed by TrkA⁺ nociceptive neurons. Doublelabeling technique was used to colocalize TrkA (green; [b,e]) and mrgs(red; [a,d]) in DRG neurons. During the double labeling experimentsfrozen sections of wild-type DRG were undergone in situ hybridizationswith either mrg3 (a-c) or mrg5 (d-f) fluorescein-labeled antisense RNAprobes followed by anti-TrkA antibody immunostaining. The same twoframes (a and b, d and e) were digitally superimposed to reveal theextent of colocalization (c, f). The colocalizations of TrkA with eithermrg3 or mrg5 appear yellow in merged images (c, f, respectively). Thewhite arrowheads indicate examples of double positive cells.

In FIG. 2C, mrgs and VR1 define two different populations of nociceptiveneurons in DRG. The combination of in situ hybridizations (red) witheither mrg3 or mrg5 fluorescein-labeled antisense RNA probes andanti-VR1 antibody immunostaining (green) demonstrated that neither mrg3(a-c) nor mrg5 (d-f) were expressed by VR1-positive neurons. In themerged images (c,f), there are no colocalizations of VR1 with eithermrg3 or mrg5. The white arrowheads are pointed to mrgs-expressing butVR1-negative nociceptive neurons.

In FIG. 2D mrgs are shown to be expressed by IB4⁺ nociceptive neurons.Double labeling technique was used to colocalize IB4 (green; [b,e]) andmrgs (red; [a,d]) in DRG neurons. The expressions of mrg3 and mrg5 werevisualized by in situ hybridization as described before. The same DRGsections were subsequently undergone through FITC-conjugated lectin IB4binding. In the merged images (c,f), there are extensive overlappingsbetween mrgs and IB4 stainings (yellow neurons indicated by arrowheads).

Information about the expression patterns of the receptors of theinvention in normal tissue and tissue taken from animal models ofdisease or patients suffering from a disease or disorder will be usefulin further defining the biological function of the receptors and intailoring treatment regimens to the specific receptor or combination ofreceptors involved in a particular disease or disorder.

I. Methods to Identify Binding Partners

As discussed in more detail below, several peptides have been putativelyidentified as endogenous ligands for Mrg receptors. In particular theRF-amide peptides, including NPAF and NPFF, have been shown toefficiently stimulate several of the Mrg receptors. In order to identifyadditional new ligands for the Mrg receptors and ligands for drg-12, itis first necessary to indentify compounds that bind to these receptors.Thus, another embodiment of the present invention provides methods ofisolating and identifying binding partners or ligands of proteins of theinvention. Macromolecules that interact with Mrg are referred to, forpurposes of this discussion, as “binding partners.” While the discussionbelow is specificially directed to identifying binding partners for Mrgreceptors, it is contemplated that the assays of the invention may beused to identify binding partners for drg-12 as well.

Receptor binding can be tested using Mrg receptors isolated from theirnative source or synthesized directly. However, Mrg receptors obtainedby the recombinant methods described above are preferred.

The compounds which may be screened in accordance with the inventioninclude, but are not limited to polypeptides, peptides, including butnot limited to members of random peptide libraries; (see, e.g., Lam, K.S. et al., 1991, Nature 354:82-84; Houghten, R. et al., 1991, Nature354:84-86) and combinatorial chemistry-derived molecular libraries madeof D- and/or L-configuration amino acids, phosphopeptides (including,but not limited to members of random or partially degenerate, directedphosphopeptide libraries; see, e.g., Songyang, Z. et al., 1993, Cell72:767-778), peptide mimetics, antibodies (including, but not limitedto, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric orsingle chain antibodies, FAb, F(abN)₂ and FAb expression libraryfragments, and epitope-binding fragments thereof), and small organic orinorganic molecules.

The ability of candidate or test compounds to bind Mrg receptors can bemeasured directly or indirectly, such as in competitive binding assays.In competitive binding experiments, the concentration of the testcompound necessary to displace 50% of another compound bound to thereceptor (IC₅₀) is used as a measure of binding affinity. In theseexperiments the other compound is a ligand known to bind to the Mrgreceptor with high affinity, such as an RF-amide peptide.

A variety of assay formats may be employed, including biochemicalscreening assays, immunoassays, cell-based assays and protein-proteinbinding assays, all of which are well characterized in the art. In oneembodiment the assay involves anchoring the test compound onto a solidphase, adding the non-immobilized component comprising the Mrg receptor,and detecting Mrg/test compound complexes anchored on the solid phase atthe end of the reaction. In an alternative embodiment, the Mrg may beanchored onto a solid surface, and the test compound, which is notanchored. In both situations either the test compound or the Mrgreceptor is labeled, either directly or indirectly, to allow foridentification of complexes. For example, an Mrg-Ig immunoadhesin may beanchored to a solid support and contacted with one or more testcompounds.

Microtiter plates are preferably utilized as the solid phase and theanchored component may be immobilized by non-covalent or covalentattachments. Non-covalent attachment may be accomplished by simplycoating the solid surface with a solution of the protein and drying.Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface.

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for either Mrgpolypeptide, peptide or fusion protein or the test compound to anchorany complexes formed in solution, and a labeled antibody specific forthe other component of the possible complex to detect anchoredcomplexes.

In one embodiment of these methods, a protein of the invention or afragment of a protein of the invention, for instance, an extracellulardomain fragment, is mixed with one or more potential binding partners,or an extract or fraction of a cell, under conditions that allow theassociation of potential binding partners with the protein of theinvention. After mixing, peptides, polypeptides, proteins or othermolecules that have become associated with a protein of the inventionare separated from the mixture. The binding partner that bound to theprotein of the invention can then be removed, identified and furtheranalyzed. To identify and isolate a binding partner, the entire Mrgprotein, for instance a protein comprising the entire amino acidsequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 21, 23, 25, 27, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107 or 109 can be used. Alternatively, a fragment of the Mrgpolypeptide can be used.

As used herein, a cellular extract refers to a preparation or fractionwhich is made from a lysed or disrupted cell. The preferred source ofcellular extracts will be cells derived from DRG. Alternatively,cellular extracts may be prepared from cells derived from any tissue,including normal human kidney tissue, or available cell lines,particularly kidney derived cell lines.

A variety of methods can be used to obtain an extract of a cell. Cellscan be disrupted using either physical or chemical disruption methods.Examples of physical disruption methods include, but are not limited to,sonication and mechanical shearing. Examples of chemical lysis methodsinclude, but are not limited to, detergent lysis and enzyme lysis. Askilled artisan can readily adapt methods for preparing cellularextracts in order to obtain extracts for use in the present methods.

Once an extract of a cell is prepared, the extract is mixed with theprotein of the invention under conditions in which association of theprotein with the binding partner can occur. Alternatively, one or moreknown compounds or molecules can be mixed with the protein of theinvention. A variety of conditions can be used, the most preferred beingconditions that closely resemble conditions found in the cytoplasm of ahuman cell. Features such as osmolarity, pH, temperature, and theconcentration of cellular extract used, can be varied to optimize theassociation of the protein with the binding partner.

After mixing under appropriate conditions, the bound complex isseparated from the mixture. A variety of techniques can be utilized toseparate the mixture. For example, antibodies specific to a protein ofthe invention can be used to immunoprecipitate the binding partnercomplex. Alternatively, standard chemical separation techniques such aschromatography and density/sediment centrifugation can be used.

After removal of non-associated cellular constituents found in theextract, and/or unbound compounds or molecules, the binding partner canbe dissociated from the complex using conventional methods. For example,dissociation can be accomplished by altering the salt concentration orpH of the mixture.

To aid in separating associated binding partner pairs from the mixedextract, the protein of the invention can be immobilized on a solidsupport. For example, the protein can be attached to a nitrocellulosematrix or acrylic beads. Attachment of the protein to a solid supportaids in separating peptide/binding partner pairs from other constituentsfound in the extract. The identified binding partners can be either asingle protein or a complex made up of two or more proteins or any othermacromolecule.

Alternatively, binding partners may be identified using a Far-Westernassay according to the procedures of Takayama et al. Methods Mol. Biol.69:171-84 (1997) or Sauder et al. J Gen. Virol. 77(5): 991-6 oridentified through the use of epitope tagged proteins or GST fusionproteins.

Binding partners may also be identified in whole cell binding assaysthat are well known in the art. In one embodiment, an Mrg receptor isexpressed in cells in which it is not normally expressed, such as COScells. The cells expressing Mrg are then contacted with a potentialbinding partner that has previously been labeled, preferably withradioactivity or a fluorescent marker. The cells are then washed toremove unbound material and the binding of the potential binding partnerto the cells is assessed, for example by collecting the cells on afilter and counting radioactivity. The amount of binding of thepotential binding partner to untransfected cells or mock transfectedcells is subtracted as background.

This type of assay may be carried out in several alternative ways. Forexample, in one embodiment it is done using cell membrane fractions fromcells transfected with an Mrg or known to express an Mrg, rather thanwhole cells. In another embodiment purified Mrg is refolded in lipids toproduce membranes that are used in the assay.

Alternatively, the nucleic acid molecules of the invention can be usedin cell based systems to detect protein-protein interactions (seeWO99/55356). These systems have been used to identify other proteinpartner pairs and can readily be adapted to employ the nucleic acidmolecules herein described.

Any method suitable for detecting protein-protein interactions may beemployed for identifying proteins, including but not limited to soluble,transmembrane or intracellular proteins, that interact with Mrgreceptors. Among the traditional methods which may be employed areco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns to identify proteins that interactwith Mrg. For such assays, the Mrg component can be a full-lengthprotein, a soluble derivative thereof, a peptide corresponding to adomain of interest, or a fusion protein containing some region of Mrg.

Methods may be employed which result in the simultaneous identificationof genes that encode proteins capable of interacting with Mrg. Thesemethods include, for example, probing expression libraries, usinglabeled Mrg or a variant thereof.

One method of detecting protein interactions in vivo that may be used toidentify Mrg binding partners is the yeast two-hybrid system. Thissystem is well known in the art and is commercially available fromClontech (Palo Alto, Calif.).

Briefly, two hybrid proteins are employed, one comprising theDNA-binding domain of a transcription activator protein fused to the Mrgreceptor, or a polypeptide, peptide, or fusion protein therefrom, andthe other comprising the transcription activator protein's activationdomain fused to an unknown target protein. These proteins are expressedin a strain of the yeast Saccharomyces cerevisiae that contains areporter gene (e.g., HBS or lacZ) whose regulatory region contains thetranscription activator's binding site. While either hybrid proteinalone cannot activate transcription of the reporter gene, interaction ofthe two hybrid proteins reconstitutes the functional activator proteinand results in expression of the reporter gene, which is detected by anassay for the reporter gene product.

The target protein is preferably obtained from tissue or cells known toexpress the Mrg receptor, such as DRG cells. For example, a cDNA libraryprepared from DRG cells may be used.

Binding partners may also be identified by their ability to interferewith or disrupt the interaction of known ligands. Even if they do notactivate Mrg receptors, binding partners that interfere withinteractions with known ligands may be useful in regulating oraugmenting Mrg activity in the body and/or controlling disordersassociated with Mrg activity (or a deficiency thereof).

Compounds that interfere with the interaction between Mrg and a knownligand may be identified by preparing a reaction mixture containing Mrg,or some variant or fragment thereof, and a known binding partner, suchas an RF-amide peptide, under conditions and for a time sufficient toallow the two to interact and bind, thus forming a complex. In order totest a compound for inhibitory activity, the reaction mixture isprepared in the presence and absence of the test compound. The testcompound may be initially included in the reaction mixture, or may beadded at a time subsequent to the addition of the Mrg and its bindingpartner. Control reaction mixtures are incubated without the testcompound. The formation of any complexes between the Mrg and the bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compoundindicates that the compound interferes with the interaction of the Mrgand the known binding partner. Additionally, complex formation withinreaction mixtures containing the test compound and normal Mrg proteinmay also be compared to complex formation within reaction mixturescontaining the test compound and a mutant Mrg. This comparison may beimportant in those cases wherein it is desirable to identify compoundsthat specifically disrupt interactions of mutant, or mutated, Mrg butnot the normal proteins.

The order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction by competition can beidentified by conducting the binding reaction in the presence of thetest substance. In this case the test compound is added to the reactionmixture prior to, or simultaneously with, Mrg and the known bindingpartner. Alternatively, test compounds that have the ability to disruptpreformed complexes can be identified by adding the test compound to thereaction mixture after complexes have been formed.

In an alternate embodiment of the invention, a preformed complex of Mrgand an interactive binding partner is prepared in which either the Mrgor its binding partners is labeled, but the signal generated by thelabel is quenched due to formation of the complex (see, e.g., U.S. Pat.No. 4,109,496 to Rubenstein which utilizes this approach forimmunoassays). The addition of a test compound that competes with anddisplaces one of the species from the preformed complex results in thegeneration of a signal above background. In this way, test substanceswhich disrupt the interaction can be identified. Whole cells expressingMrg, membrane fractions prepared from cells expressing Mrg or membranescontaining refolded Mrg may be used in the assays described above.However, these same asays can be employed using peptide fragments thatcorrespond to the binding domains of Mrg and/or the interactive orbinding partner (in cases where the binding partner is a protein), inplace of one or both of the full length proteins. Any number of methodsroutinely practiced in the art can be used to identify and isolate thebinding sites. These methods include, but are not limited to,mutagenesis of the gene encoding an Mrg protein and screening fordisruption of binding of a known ligand.

The compounds identified can be useful, for example, in modulating theactivity of wild type and/or mutant Mrg; can be useful in elaboratingthe biological function of Mrg receptors; can be utilized in screens foridentifying compounds that disrupt normal Mrg receptor interactions ormay themselves disrupt or activate such interactions; and can be usefultherapeutically.

J. Methods to Identify Agents that Modulate the Expression of a NucleicAcid.

Another embodiment of the present invention provides methods foridentifying agents that modulate the expression of a nucleic acidencoding a mrg or drg-12 protein of the invention or another proteininvolved in an mrg or drg-12 mediated pathway. These agents may be, butare not limited to, peptides, peptide mimetics, and small organicmolecules that are able to gain entry into an appropriate cell (e.g., inthe DRG) and affect the expression of a gene. Agents that modulate theexpression of Mrg or drg-12 or a protein in an mrg mediated pathway maybe useful therapeutically, for example to increase or decrease sensoryperception, such as the perception of pain, to treat glaucoma, or toincrease or decrease wound healing.

Such assays may utilize any available means of monitoring for changes inthe expression level of the nucleic acids of the invention. As usedherein, an agent is said to modulate the expression of a nucleic acid ofthe invention, for instance a nucleic acid encoding the protein havingthe sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107 or 109 if it is capable of up- ordown-regulating expression of the gene or mRNA levels nucleic acid in acell.

In one assay format, cell lines that contain reporter gene fusionsbetween the open reading frames and/or the 5′ or 3′ regulatory sequencesof a gene of the invention and any assayable fusion partner may beprepared. Numerous assayable fusion partners are known and readilyavailable including the firefly luciferase gene and the gene encodingchloramphenicol acetyltransferase (Alam et al. Anal. Biochem.188:245-254 (1990)). Cell lines containing the reporter gene fusions arethen exposed to the agent to be tested under appropriate conditions andtime. Differential expression of the reporter gene between samplesexposed to the agent and control samples identifies agents whichmodulate the expression of a nucleic acid encoding a mrg or drg-12protein.

Additional assay formats may be used to monitor the ability of the agentto modulate the expression of a nucleic acid encoding a mrg or drg-12protein of the invention. For instance, mRNA expression may be monitoreddirectly by hybridization to the nucleic acids of the invention. Celllines are exposed to the agent to be tested under appropriate conditionsand time and total RNA or mRNA is isolated by standard procedures suchthose disclosed in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2nd Ed. Cold Spring Harbor Laboratory Press, 1989).

Probes to detect differences in RNA expression levels between cellsexposed to the agent and control cells may be prepared from the nucleicacids of the invention. It is preferable, but not necessary, to designprobes which hybridize only with target nucleic acids under conditionsof high stringency. Only highly complementary nucleic acid hybrids formunder conditions of high stringency. Accordingly, the stringency of theassay conditions determines the amount of complementarity which shouldexist between two nucleic acid strands in order to form a hybrid.Stringency should be chosen to maximize the difference in stabilitybetween the probe:target hybrid and potential probe:non-target hybrids.

Probes may be designed from the nucleic acids of the invention throughmethods known in the art. For instance, the G+C content of the probe andthe probe length can affect probe binding to its target sequence.Methods to optimize probe specificity are commonly available in Sambrooket al. (Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold SpringHarbor Laboratory Press, NY, 1989) or Ausubel et al. (Current Protocolsin Molecular Biology, Greene Publishing Co., NY, 1995).

Hybridization conditions are modified using known methods, such as thosedescribed by Sambrook et al. and Ausubel et al., as required for eachprobe. Hybridization of total cellular RNA or RNA enriched for polyA RNAcan be accomplished in any available format. For instance, totalcellular RNA or RNA enriched for polyA RNA can be affixed to a solidsupport and the solid support exposed to at least one probe comprisingat least one, or part of one of the sequences of the invention underconditions in which the probe will specifically hybridize.Alternatively, nucleic acid fragments comprising at least one, or partof one of the sequences of the invention can be affixed to a solidsupport, such as a silicon chip or porous glass wafer. The wafer canthen be exposed to total cellular RNA or polyA RNA from a sample underconditions in which the affixed sequences will specifically hybridize.Such wafers and hybridization methods are widely available, for example,those disclosed by Beattie (WO 95/11755). By examining for the abilityof a given probe to specifically hybridize to an RNA sample from anuntreated cell population and from a cell population exposed to theagent, agents which up or down regulate the expression of a nucleic acidencoding a mrg or drg-12 are identified.

Hybridization for qualitative and quantitative analysis of mRNAs mayalso be carried out by using a RNase Protection Assay (i.e., RPA, see Maet al. Methods 10: 273-238 (1996)). Briefly, an expression vehiclecomprising cDNA encoding the gene product and a phage specific DNAdependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase)is linearized at the 3′ end of the cDNA molecule, downstream from thephage promoter, wherein such a linearized molecule is subsequently usedas a template for synthesis of a labeled antisense transcript of thecDNA by in vitro transcription. The labeled transcript is thenhybridized to a mixture of isolated RNA (i.e., total or fractionatedmRNA) by incubation at 45° C. overnight in a buffer comprising 80%formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA. The resultinghybrids are then digested in a buffer comprising 40 μg/ml ribonuclease Aand 2 μg/ml ribonuclease. After deactivation and extraction ofextraneous proteins, the samples are loaded onto urea/polyacrylamidegels for analysis.

In another assay format, products, cells or cell lines are first beidentified which express mrg or drg-12 gene products physiologically.Cells and/or cell lines so identified would be expected to comprise thenecessary cellular machinery such that the fidelity of modulation of thetranscriptional apparatus is maintained with regard to exogenous contactof agent with appropriate surface transduction mechanisms and/or thecytosolic cascades. Such cells or cell lines are then transduced ortransfected with an expression vehicle (e.g., a plasmid or viral vector)construct comprising an operable non-translated 5′ or 3′-promotercontaining end of the structural gene encoding the instant gene productsfused to one or more antigenic fragments, which are peculiar to theinstant gene products, wherein said fragments are under thetranscriptional control of said promoter and are expressed aspolypeptides whose molecular weight can be distinguished from thenaturally occurring polypeptides or may further comprise animmunologically distinct tag. Such a process is well known in the art.

Cells or cell lines transduced or transfected as outlined above are thencontacted with agents under appropriate conditions; for example, theagent comprises a pharmaceutically acceptable excipient and is contactedwith cells comprised in an aqueous physiological buffer such asphosphate buffered saline (PBS) at physiological pH, Eagles balancedsalt solution (BSS) at physiological pH, PBS or BSS comprising serum orconditioned media comprising PBS or BSS and/or serum incubated at 37° C.Said conditions may be modulated as deemed necessary by one of skill inthe art. Subsequent to contacting the cells with the agent, said cellswill be disrupted and the polypeptides of the lysate are fractionatedsuch that a polypeptide fraction is pooled and contacted with anantibody to be further processed by immunological assay (e.g., ELISA,immunoprecipitation or Western blot). The pool of proteins isolated fromthe “agent-contacted” sample will be compared with a control samplewhere only the excipient is contacted with the cells and an increase ordecrease in the immunologically generated signal from the“agent-contacted” sample compared to the control will be used todistinguish the effectiveness of the agent.

The probes described above for identifying differential expression ofMrg mRNA in response to applied agents can also be used to identifydifferential expression of Mrg mRNA in populations of mammals, forexample populations with differing levels of sensory perception. Methodsfor identifying differential expression of genes are well known in theart. In one embodiment, mRNA is prepared from tissue or cells taken frompatients exhibiting altered sensory perception, such as patientsexperiencing neuropathic pain, or suffering from a disease or disorderin which the Mrg receptor may play a role, such as glaucoma, and Mrgexpression levels are quantified using the probes described above. TheMrg expression levels may then be compared to those in other populationsto determine the role that Mrg expression is playing in the alterationof sensory perception and to determine whether treatment aimed atincreasing or decreasing Mrg expression levels would be appropriate.

K. Methods to Identify Agents that Modulate Protein Levels or at LeastOne Activity of the Proteins of DRG Primary Sensory Neurons.

Another embodiment of the present invention provides methods foridentifying agents or conditions that modulate protein levels and/or atleast one activity of a mrg or drg-12 protein of the invention,including agonists and antagonists. Such methods or assays may utilizeany means of monitoring or detecting the desired activity.

In one format, the relative amounts of a protein of the inventionbetween a cell population that has been exposed to the agent to betested compared to an unexposed control cell population may be assayed.In this format, probes such as specific antibodies are used to monitorthe differential expression of the protein in the different cellpopulations. Cell lines or populations are exposed to the agent to betested under appropriate conditions and time. Cellular lysates may beprepared from the exposed cell line or population and a control,unexposed cell line or population. The cellular lysates are thenanalyzed with the probe.

In another embodiment, animals known to express Mrg or drg-12 receptorsare subjected to a particular environmental stimulus and any changeproduced in Mrg or drg-12 protein expression by exposure to the stimulusis measured. Transgenic animals, such as transgenic mice, produced toexpress a particular Mrg in a particular location may be used. Theenvironmental stimulus is not limited and may be, for example, exposureto stressful conditions, or exposure to noxious or painful stimuli.Differences in Mrg receptor expression levels in response toenvironmental stimuli may provide insight into the biological role ofMrgs and possible treatments for diseases or disorders related to thestimuli used.

Antibody probes are prepared by immunizing suitable mammalian hosts inappropriate immunization protocols using the peptides, polypeptides orproteins of the invention if they are of sufficient length, or, ifdesired, or if required to enhance immunogenicity, conjugated tosuitable carriers. Methods for preparing immunogenic conjugates withcarriers such as BSA, KLH, or other carrier proteins are well known inthe art. In some circumstances, direct conjugation using, for example,carbodiimide reagents may be effective; in other instances linkingreagents such as those supplied by Pierce Chemical Co. (Rockford, Ill.),may be desirable to provide accessibility to the hapten. The haptenpeptides can be extended at either the amino or carboxy terminus with acysteine residue or interspersed with cysteine residues, for example, tofacilitate linking to a carrier. Administration of the immunogens isconducted generally by injection over a suitable time period and withuse of suitable adjuvants, as is generally understood in the art. Duringthe immunization schedule, titers of antibodies are taken to determineadequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactoryfor some applications, for pharmaceutical compositions, use ofmonoclonal preparations is preferred. Immortalized cell lines whichsecrete the desired monoclonal antibodies may be prepared using thestandard method of Kohler and Milstein Nature 256:495-497 (1975)) ormodifications which effect immortalization of lymphocytes or spleencells, as is generally known. The immortalized cell lines secreting thedesired antibodies are screened by immunoassay in which the antigen isthe peptide hapten, polypeptide or protein. When the appropriateimmortalized cell culture secreting the desired antibody is identified,the cells can be cultured either in vitro or by production in ascitesfluid.

The desired monoclonal antibodies are then recovered from the culturesupernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab′, of F(ab′)₂ fragments is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Antibody regions that bindspecifically to the desired regions of the protein can also be producedin the context of chimeras with multiple species origin, such ashumanized antibodies as discussed in more detail below.

1. Identification of Agonists and Antagonists

The present invention provides for assays to identify compounds thatserve as agonists or antagonists of one or more of the biologicalproperties of Mrg and/or drg-12. Mrg agonists and antagonists may beuseful in the prevention and treatment of problems associated withsensory perception, particularly nociception. Mrg agonists andantagonists may alter sensory perception, particularly the perception ofpain. For example, compounds identified as Mrg receptor agonists may beused to stimulate Mrg receptor activation and thus may be effective intreating mammals suffering from pain by reducing the perception of pain.Compounds that are identified as Mrg receptor antagonists may be used,for example, to decrease the effector functions of Mrg receptors. Thismay be useful in cases where the Mrg receptors contain a mutation thatproduces increased responsiveness, or in cases of Mrg receptoroverexpression. For instance, Mrg receptor antagonists may be useful inincreasing the sensitivity of mammals to pain where appropriate, such asin diseases involving decreased sensory responsiveness, like some formsof diabetes.

Assays for identifying agonists or antagonsts may be done in vitro or invivo, by monitoring the response of a cell following binding of theligand to the receptor. An agonist will produce a cellular response,while an antagonist will have no effect on cellular response but will becapable of preventing cellular response to a known agonist.

a. Small Molecules

Small molecules may have the ability to act as Mrg agonists orantagonists and thus may be screened for an effect on a biologicalactivity of Mrg. Small molecules preferably have a molecular weight ofless than 10 kD, more preferably less than 5 kD and even more preferablyless than 2 kD. Such small molecules may include naturally occurringsmall molecules, synthetic organic or inorganic compounds, peptides andpeptide mimetics. However, small molecules in the present invention arenot limited to these forms. Extensive libraries of small molecules arecommercially available and a wide variety of assays are well known inthe art to screen these molecules for the desired activity.

Candidate Mrg agonist and antagonist small molecules are preferablyfirst identified in an assay that allows for the rapid identification ofpotential agonists and antagonists. An example of such an assay is abinding assay wherein the ability of the candidate molecule to bind tothe Mrg receptor is measured, such as those described above. In anotherexample, the ability of candidate molecules to interfere with thebinding of a known ligand, for example FMRFamide to MrgA1, is measured.Candidate molecules that are identified by their ability to bind to Mrgproteins or interfere with the binding of known ligands are then testedfor their ability to stimulate one or more biological activities.

The activity of the proteins of the invention may be monitored in cellsexpressing the mrg and/or drg-12 proteins of the invention by assayingfor physiological changes in the cells upon exposure to the agent oragents to be tested. Such physiological changes include but are notlimited to the flow of current across the membrane of the cell.

In one embodiment the protein is expressed in a cell that is capable ofproducing a second messenger response and that does not normally expressMrg or drg-12. The cell is then contacted with the compound of interestand changes in the second messenger response are measured. Methods tomonitor or assay these changes are readily available. For instance, themrg genes of the invention may be expressed in cells expressing Gα15, aG protein α subunit that links receptor activation to increases inintracellular calcium [Ca²⁺] which can be monitored at the single celllevel using the FURA-2 calcium indicator dye as disclosed inChandrashekar et al. Cell 100:703-711, (2000). This assay is describedin more detail in Example 5.

Similar assays may also be used to identify inhibitors or antagonists ofMrg or drg-12 activation. For example, cells expressing Mrg or drg-12and capable of producing a quantifiable response to receptor activationare contacted with a known Mrg or drg-12 activator and the compound tobe tested. In one embodiment, HEK cells expressing Gα15 and MrgA1 arecontacted with FMRFamide and the compound to be tested. The cellularresponse is measured, in this case increase in [Ca²⁺]. A decreasedresponse compared to the known activator by itself indicates that thecompound acts as an inhibitor of activation.

While such assays may be formatted in any manner, particularly preferredformats are those that allow high-throughput screening (HTP). In HTPassays of the invention, it is possible to screen thousands of differentmodulators or ligands in a single day. For instance, each well of amicrotiter plate can be used to run a separate assay, for instance anassay based on the ability of the test compounds to modulate receptoractivation derived increases in intracellular calcium as describedabove.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences involved in the association of the a protein ofthe invention alone or with its associated substrates, binding partners,etc. An example of randomly selected agents is the use a chemicallibrary or a peptide combinatorial library, or a growth broth of anorganism.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis which takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. Sites of interest might be peptides within themembrane spanning regions, cytoplasmic and extracellular peptide loopsbetween these transmembrane regions, or selected sequences within theN-terminal extracellular domain or C-terminal intracellular domain.Agents can be rationally selected or rationally designed by utilizingthe peptide sequences that make up these sites.

The agents of the present invention can be, as examples, peptides, smallmolecules, vitamin derivatives, as well as carbohydrates. Dominantnegative proteins, DNAs encoding these proteins, antibodies to theseproteins, peptide fragments of these proteins or mimics of theseproteins may be introduced into cells to affect function. “Mimic” usedherein refers to the modification of a region or several regions of apeptide molecule to provide a structure chemically different from theparent peptide but topographically and functionally similar to theparent peptide (see Grant G A. in: Meyers (ed.) Molecular Biology andBiotechnology (New York, VCH Publishers, 1995), pp. 659-664). A skilledartisan can readily recognize that there is no limit as to thestructural nature of the agents of the present invention.

The peptide agents of the invention can be prepared using standard solidphase (or solution phase) peptide synthesis methods, as is known in theart. In addition, the DNA encoding these peptides may be synthesizedusing commercially available oligonucleotide synthesis instrumentationand produced recombinantly using standard recombinant productionsystems. The production using solid phase peptide synthesis isnecessitated if non-gene-encoded amino acids are to be included.

b. Antibodies

Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of proteins of the invention.These antibodies may be human or non-human, polyclonal or monoclonal andmay serve as agonist antibodies or neutralizing antibodies. They includeamino acid sequence variants, glycosylation variants and fragments ofantibodies. Antibody agents are obtained by immunization of suitablemammalian subjects with peptides, containing as antigenic regions, thoseportions of the protein intended to be targeted by the antibodies.General techniques for the production of such antibodies and theselection of agonist or neutralizing antibodies are well known in theart.

The antibodies of the present invention can be polyclonal antibodies,monoclonal antibodies, chimeric antibodies, humanized antibodies, humanantibodies, heteroconjugate antibodies, or antibody fragments. Inaddition, the antibodies can be made by any method known in the art,including recombinant methods.

Mrg agonist and neutralizing antibodies may be preliminarily identifiedbased on their ability to bind the Mrg receptor. For example, Westernblot techniques well known in the art may be used to screen a variety ofantibodies for their ability to bind Mrg. Mrg agonist and neutralizingantibodies are then identified from the group of candidate antibodiesbased on their biological activity. In one embodiment, Mrg agonistantibodies are identified by their ability to induce activation of asecond messenger system in cells expressing the Mrg protein andcomprising a second messenger system. For example, HEK cellsoverexpressing Gα15 and transfected with mrg may be contacted with apotential Mrg agonist antibody. An increase in intracellular calcium,measured as described in Example 5, would indicate that the antibody isan agonist antibody.

Identification of a neutralizing antibody involves contacting a cellexpressing Mrg with a known Mrg ligand, such as an RF-amide peptide, andthe candidate antibody and observing the effect of the antibody on Mrgactivation. In one embodiment, Mrg receptors expressed in HEK cellsoverexpressing Gα15 are contacted with an Mrg ligand such as FMRFamideand the candidate neutralizing antibody. A decrease in responsiveness tothe ligand, measured as described in Example 5, would indicate that theantibody is a neutralizing antibody.

c. Other Antagonists

The Mrg or drg-12 antagonists are not limited to Mrg or drg-12 ligands.Other antagonists include variants of a native Mrg or drg-12 receptorthat retains the ability to bind an endogenous ligand but is not able tomediate a biological response. Soluble receptors and immunoadhesins thatbind Mrg or drg-12 ligands may also be antagonists, as may antibodiesthat specifically bind a ligand near its binding site and prevent itsinteraction with the native receptor. These antagonists may beidentified in the assays described above.

d. Computer Modeling

Computer modeling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate Mrg receptor expression or activity. Once an agonist orantagonist is identified, the active sites or regions, such as ligandbinding sites, are determined. The active site can be identified usingmethods known in the art including, for example, by determining theeffect of various amino acid substitutions or deletions on ligandbinding or from study of complexes of the relevant compound orcomposition with its natural ligand, such as with X-ray crystallography.

Next, the three dimensional geometric structure of the active site isdetermined such as by X-ray crystallography, NMR, chemical crosslinkingor other methods known in the art. Computer modeling can be utilized tomake predictions about the structure where the experimental results arenot clear. Examples of molecular modeling systems are the CHARMm andQUANTA programs (Polygen Corporation, Waltham, Mass.). Once a predictedstructure is determined, candidate modulating compounds can beidentified by searching databases containing compounds along withinformation on their molecular structure in an effort to find compoundsthat have structures capable of interacting with the active site. Thecompounds found from this search are potential modulators of theactivity of the proteins of the present invention and can be tested inthe assays described above.

The agonistic or antagonistic activity of test compounds identified incell based assays as described above can be further elucidated in assaysusing animals, for example transgenic animals that overexpress Mrgreceptors as described in more detail below. In one embodiment, theeffect of administration of potential Mrg antagonists or agonists on theresponsiveness of such transgenic animals to sensory stimuli, such asnoxious or painful stimuli, is measured. The therapeutic utility of suchcompounds may be confirmed by testing in these types of experiments orin animal models of particular disorders, for example animal models ofneuropathic pain.

L. Uses for Agents that Modulate at Least One Activity of the Proteins.

As provided in the Examples, the mrg or drg-12 proteins and nucleicacids of the invention, are expressed in the primary nociceptive sensoryneurons of DRG. In addition the Mrg receptors are expressed inspecialized skin cells that play a role in wound repair. Further,proteins homologous to Mrg receptors are expressed in the trabecularmeshwork of the eye and a role for them has been suggested in theregulation of pressure in the eye (Gonzalez et al. Invest. Ophth. Vis.Sci. 41: 3678-3693 (2000)). Thus, the present invention further providescompositions containing one or more agents that modulate expression orat least one activity of a protein of the invention. For example, theinvention provides ligands that directly activate Mrg receptors.

Agents that modulate, up-or-down-regulate the expression of the proteinor agents such as agonists or antagonists of at least one activity ofthe protein may be used to modulate biological and pathologic processesassociated with the protein's function and activity. Several agents thatactivate the Mrg receptors are identified in the examples, including theRF-amide peptides. Thus the present invention provides methods to treatimpaired sensory perception, such as pain, including neuropathic pain,as well as to promote wound healing, to restore normal sensitivityfollowing injury and to treat ocular conditions, particularly thoseassociated with pressure, such as glaucoma.

As described in the Figures and Examples, expression of a protein of theinvention may be associated with biological processes of nociception,which may also be considered pathological processes. As used herein, anagent is said to modulate a biological or pathological process when theagent alters the degree, severity or nature of the process. Forinstance, the neuronal transmission of pain signals may be prevented ormodulated by the administration of agents which up-regulatedown-regulate or modulate in some way the expression or at least oneactivity of a protein of the invention.

The pain that may be treated by the proteins of the present inventionand agonists and antagonists thereof, is not limited in any way andincludes pain associated with a disease or disorder, pain associatedwith tissue damage, pain associated with inflammation, pain associatedwith noxious stimuli of any kind, and neuropathic pain, including painassociated with peripheral neuropathies, as well as pain without anidentifiable source. The pain may be subjective and does not have to beassociated with an objectively quantifiable behavior or response.

In addition to treating pain, the compounds and methods of the presentinvention may be useful for increasing or decreasing sensory responses.It may be useful to increase responsiveness to stimuli, includingnoxious stimuli and painful stimuli, in some disease states that arecharacterized by a decreased responsiveness to stimuli, for example indiabetes.

Certain conditions, such as chronic disease states associated with painand peripheral neuropathies and particularly conditions resulting from adefective Mrg gene, can benefit from an increase in the responsivenessto Mrg receptor ligands. Thus these condition may be treated byincreasing the number of functional Mrg receptors in cells of patientssuffering from such conditions. This could be increasing the expressionof Mrg receptor in cells through gene therapy using Mrg-encoding nucleicacid. This includes both gene therapy where a lasting effect is achievedby a single treatment, and gene therapy where the increased expressionis transient. Selective expression of Mrg in appropriate cells may beachieved by using Mrg genes controlled by tissue specific or induciblepromoters or by producing localized infection with replication defectiveviruses carrying a recombinant Mrg gene, or by any other method known inthe art.

In a further embodiment, patients that suffer from an excess of Mrg,hypersensitivity to Mrg ligands or excessive activation of Mrg may betreated by administering an effective amount of anti-sense RNA oranti-sense oligodeoxyribonucleotides corresponding to the Mrg genecoding region, thereby decreasing expression of Mrg.

As used herein, a subject to be treated can be any mammal, so long asthe mammal is in need of modulation of a pathological or biologicalprocess mediated by a protein of the invention. For example, the subjectmay be experiencing pain or may be anticipating a painful event, such assurgery. The invention is particularly useful in the treatment of humansubjects.

In the therapeutic methods of the present invention the patient isadministered an effective amount of a composition of the presentinvention, such as an Mrg protein, peptide fragment, Mrg variant, Mrgagonist, Mrg antagonist, or anti-Mrg antibody of the invention.

The agents of the present invention can be provided alone, or incombination with other agents that modulate a particular biological orpathological process. For example, an agent of the present invention canbe administered in combination with other known drugs or may be combinedwith analgesic drugs or non-analgesic drugs used during the treatment ofpain that occurs in the presence or absence of one or more otherpathological processes. As used herein, two or more agents are said tobe administered in combination when the two agents are administeredsimultaneously or are administered independently in a fashion such thatthe agents will act at the same time.

The agents of the present invention are administered to a mammal,preferably to a human patient, in accord with known methods. Thus theagents of the present invention can be administered via parenteral,subcutaneous, intravenous, intramuscular, intraperitoneal,intracerebrospinal, intra-articular, intrasynovial, intrathecal,transdermal, topical, inhalation or buccal routes. They may beadministered continuously by infusion or by bolus injection. Generally,where the disorder permits the agents should be delivered in asite-specific manner. Alternatively, or concurrently, administration maybe by the oral route. The dosage administered will be dependent upon theage, health, and weight of the recipient, kind of concurrent treatment,if any, frequency of treatment, and the nature of the effect desired.

The toxicity and therapeutic efficacy of agents of the present inventioncan be determined by standard pharmaceutical procedures in cell culturesor experimental animals. While agents that exhibit toxic side effectscan be used, care should be taken to design a delivery system thattargets such compounds to the desired site of action in order to reduceside effects.

While individual needs vary, determination of optimal ranges ofeffective amounts of each component is within the skill of the art. Forthe prevention or treatment of disease, the appropriate dosage of agentwill depend on the type of disease to be treated, the severity andcourse of the disease, whether the agent is administered for preventiveor therapeutic purposes, previous therapy, the patient's clinicalhistory and response to the agent, and the discretion of the attendingphysician. Therapeutic agents are suitably administered to the patientat one time or over a series of treatments. Typical dosages comprise 0.1to 100 μg/kg body wt. The preferred dosages comprise 0.1 to 10 μg/kgbody wt. The most preferred dosages comprise 0.1 to 1 μg/kg body wt. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. The progress of this therapy is easilymonitored by conventional techniques and assays.

In addition to the pharmacologically active agent, the compositions ofthe present invention may contain suitable pharmaceutically acceptablecarriers comprising excipients and auxiliaries that facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically for delivery to the site of action. Suitableformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form, for example, water-solublesalts. In addition, suspensions of the active compounds as appropriateoily injection suspensions may be administered. Suitable lipophilicsolvents or vehicles include fatty oils, for example, sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers. Liposomes can also be used to encapsulate the agentfor delivery into the cell. The agent can also be prepared as asustained-release formulation, including semipermeable matrices of solidhydrophobic polymers containing the protein. The sustained releasepreparation may take the form of a gel, film or capsule.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for enteral, parenteral or topicaladministration. Indeed, all three types of formulations may be usedsimultaneously to achieve systemic administration of the activeingredient.

Suitable formulations for oral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release forms thereof.

In practicing the methods of this invention, the compounds of thisinvention may be used alone or in combination with other therapeutic ordiagnostic agents. In certain preferred embodiments, the compounds ofthis invention may be co-administered along with other compoundstypically prescribed for these conditions according to generallyaccepted medical practice. The compounds of this invention can beutilized in vivo, ordinarily in mammals, such as humans, sheep, horses,cattle, pigs, dogs, cats, rats and mice, or in vitro. When used in vivo,the compounds must be sterile. This is readily accomplished byfiltration through sterile filtration membranes.

a. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label or package insert(s) on or associated with the container.Suitable containers include, for example, bottles, vials, syringes, etc.The containers may be formed from a variety of materials such as glassor plastic. The container holds a composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is an Mrg agonist. The label or packageinsert indicates that the composition is used for treating the conditionof choice, such as to treat impaired sensory perception, for example toreduce neuropathic pain. In one embodiment, the label or package insertsindicates that the composition comprising the Mrg agonist can be used totreat pain, glaucoma or to accelerate wound healing.

M. Transgenic Animals

Transgenic animals containing mutant, knock-out or modified genescorresponding to the mrg and/or drg-12 sequences are also included inthe invention. Transgenic animals are genetically modified animals intowhich recombinant, exogenous or cloned genetic material has beenexperimentally transferred. Such genetic material is often referred toas a “transgene”. The nucleic acid sequence of the transgene, in thiscase a form of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 20, 22, 24, 26or 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 7274, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106 or 108 may be integrated either at a locus of agenome where that particular nucleic acid sequence is not otherwisenormally found or at the normal locus for the transgene. In addition thetransgene may encode a non-functional variant. The transgene may consistof nucleic acid sequences derived from the genome of the same species orof a different species than the species of the target animal.

The term “germ cell line transgenic animal” refers to a transgenicanimal in which the genetic alteration or genetic information wasintroduced into a germ line cell, thereby conferring the ability of thetransgenic animal to transfer the genetic information to offspring. Ifsuch offspring in fact possess some or all of that alteration or geneticinformation, then they too are transgenic animals.

The alteration or genetic information may be foreign to the species ofanimal to which the recipient belongs, foreign only to the particularindividual recipient, or may be genetic information already possessed bythe recipient. In the last case, the altered or introduced gene may beexpressed differently than the native gene.

Transgenic animals can be produced by a variety of different methodsincluding transfection, electroporation, microinjection, gene targetingin embryonic stem cells and recombinant viral and retroviral infection(see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins etal. Hypertension 22(4):630-633 (1993); Brenin et al. Surg. Oncol.6(2)99-110 (1997); Tuan (ed.), Recombinant Gene Expression Protocols,Methods in Molecular Biology No. 62, Humana Press (1997)).

A number of recombinant or transgenic mice have been produced, includingthose which express an activated oncogene sequence (U.S. Pat. No.4,736,866); express simian SV40 T-antigen (U.S. Pat. No. 5,728,915);lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Pat.No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No.5,723,719); express at least one human gene which participates in bloodpressure control (U.S. Pat. No. 5,731,489); display greater similarityto the conditions existing in naturally occurring Alzheimer's disease(U.S. Pat. No. 5,720,936); have a reduced capacity to mediate cellularadhesion (U.S. Pat. No. 5,602,307); possess a bovine growth hormone gene(Clutter et al. Genetics 143(4):1753-1760 (1996)); or, are capable ofgenerating a fully human antibody response (McCarthy The Lancet349(9049):405 (1997)).

While mice and rats remain the animals of choice for most transgenicexperimentation, in some instances it is preferable or even necessary touse alternative animal species. Transgenic procedures have beensuccessfully utilized in a variety of non-murine animals, includingsheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits,cows and guinea pigs (see, e.g., Kim et al. Mol. Reprod. Dev. 46(4):515-526 (1997); Houdebine Reprod. Nutr. Dev. 35(6):609-617 (1995);Petters Reprod. Fertil. Dev. 6(5):643-645 (1994); Schnieke et al.Science 278(5346):2130-2133 (1997); and Amoah J. Animal Science75(2):578-585 (1997)).

The method of introduction of nucleic acid fragments into recombinationcompetent mammalian cells can be by any method that favorsco-transformation of multiple nucleic acid molecules. Detailedprocedures for producing transgenic animals are readily available to oneskilled in the art, including the disclosures in U.S. Pat. No. 5,489,743and U.S. Pat. No. 5,602,307.

It is contemplated that mice lacking a particular Mrg or drg-12 gene, orin which expression of a particular Mrg or drg-12 has been increased ordecreased will be used in an assay for determining how Mrgs influencebehavior, including sensory responses, particularly responses to painfulstimuli. In particular, transgenic mice will be used to determine if Mrgmediates the response to a particular type of noxious stimuli, such asmechanical, thermal or chemical. Thus in one embodiment transgenic micelacking native Mrg receptors, or in which Mrg receptor expression levelshave been modified, will be tested to determine their sensitivity topressure, temperature, and other noxious stimuli. Assays for determiningsensitivity to stimuli are well known in the art. These include, but arenot limited to, assays that measure responsiveness to mechanical pain(von Frey hairs or tail pinch), thermal pain (latency to lick or jump inthe hot plate assay), chemical pain (latency to lick when a noxioussubstance such as capsaicin or formalin is injected in the paw),visceral pain (abdominal stretching in response to intraperitonealinjection of acetic acid) and neuropathic pain. For example, mice inwhich one or more Mrgs have been deleted will be tested for theirresponsiveness to a variety of painful stimuli of varying intensity. Bydetermining the sensory responses that are mediated by the Mrgreceptors, therapeutic agents known to stimulate or inhibit Mrgreceptors can be chosen for the treatment of disease states known toinvolve these types of responses. In addition, therapeutics specificallyaimed at treating disorders involving these responses can be developedby targeting the Mrg receptors.

In one embodiment, transgenic mice expressing one or more human Mrgproteins are produced. The expression pattern of the human Mrg proteinmay then be determined and the effect of the expression of the human Mrgprotein on various sensory modalities may be investigated. Further, theefficacy of potential therapeutic agents may be investigated in thesemice.

In addition, the effects of changes in the expression levels of specificMrg proteins can be investigated in animal models of disease states. Byidentifying the effect of increasing or decreasing Mrg receptor levelsand activation, therapeutic regimens useful in treating the diseases canbe developed. In one embodiment, mice in which Mrg receptor expressionlevels have been increased or decreased are tested in models ofneuropathic pain.

Further, mice in which Mrg expression levels have been manipulated maybe tested for their ability to respond to compounds known to modulateresponsiveness to pain, such as analgesics. In this way the role of Mrgin the sensation of pain may be further elucidated. For example, a lackof response to a known analgesic in the transgenic mice lacking Mrgwould indicate that the Mrg receptors play a role in mediating theaction of the analgesic.

Another preferred transgenic mouse is one in which the Mrg gene ismodified to express a marker or tracer such as green fluorescent protein(GFP). By examining the expression pattern of the marker or tracer, theexact location and projection of Mrg containing neurons and other cellscan be mapped. This information will be compared to the location andprojection of neurons and other cells whose involvement in specificdisease states has previously been identified. In this way additionaltherapeutic uses for the compounds of the present invention may berealized.

N. Diagnostic Methods

As described in the Examples, the genes and proteins of the inventionmay be used to diagnose or monitor the presence or absence of sensoryneurons and of biological or pathological activity in sensory neurons.For instance, expression of the genes or proteins of the invention maybe used to differentiate between normal and abnormal sensory neuronalactivities associated with acute pain, chronic intractable pain, orallodynia. Expression levels can also be used to differentiate betweenvarious stages or the severity of neuronal abnormalities. One means ofdiagnosing pathological states of sensory neurons involved in paintransmission using the nucleic acid molecules or proteins of theinvention involves obtaining tissue from living subjects. These subjectsmay be non-human animal models of pain.

The use of molecular biological tools has become routine in forensictechnology. For example, nucleic acid probes may be used to determinethe expression of a nucleic acid molecule comprising all or at leastpart of the sequences of the invention in forensic/pathology specimens.Further, nucleic acid assays may be carried out by any means ofconducting a transcriptional profiling analysis. In addition to nucleicacid analysis, forensic methods of the invention may target the proteinsof the invention to determine up or down regulation of the genes(Shiverick et al., Biochim Biophys Acta 393(1): 124-33 (1975)).

Methods of the invention may involve treatment of tissues withcollagenases or other proteases to make the tissue amenable to celllysis (Semenov et al., Biull Eksp Biol Med 104(7): 113-6 (1987)).Further, it is possible to obtain biopsy samples from different regionsof the kidney or other tissues for analysis.

Assays to detect nucleic acid or protein molecules of the invention maybe in any available format. Typical assays for nucleic acid moleculesinclude hybridization or PCR based formats. Typical assays for thedetection of proteins, polypeptides or peptides of the invention includethe use of antibody probes in any available format such as in situbinding assays, etc. See Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988 and Section G. In preferredembodiments, assays are carried-out with appropriate controls.

The above methods may also be used in other diagnostic protocols,including protocols and methods to detect disease states in othertissues or organs.

O. Methods of Identifying Other Genes Expressed in Primary NociceptiveSensory Neurons.

As described in the Examples, the mrg and drg-12 genes of the inventionhave been identified RNA using a suppression-PCR-based method (Clontech)to enrich for genes expressed in the DRG of wild type but not Ngn1mutant mice. This general method may be used to identify and isolateother DRG specific genes by producing transgenic mice that do notexpress other genes required for the development or presence of thenociceptive subset of DRG neurons. For instance, TrkA^(−/−) mice may beused in the methods of the invention to isolate other genes associatedwith nociceptive DRG neurons (see Lindsay Philos. Trans R. Soc. Lond. B.Biol. Sci. 351(1338): 365-73 (1996) and Walsh et al. J. Neurosci.19(10): 4155-68).

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure.

EXAMPLES Example 1 Positive Selection-Based Differential HybridizationBetween Wild Type and Ngn1^(−/−) DRG to Identify Candidate GenesInvolved in Nociception

Previous studies have shown that Neurogenin1 (Ngn1), a bHLHtranscription factor (Ma et al. Cell 87: 43-52 (1996)), is required forcell fate determination of nociceptive sensory neurons in dorsal rootganglia (DRG) (Ma et al. Genes & Dev. 13: 1717-1728 (1999)). InNgn1^(−/−) mutant mouse embryos most if not all trkA⁺ neurons, whichinclude the nociceptive subclass, fail to be generated. This mutantphenotype was exploited to isolate genes specifically expressed in suchneurons, by subtracting cDNAs from neonatal wild-type and Ngn1^(−/−)DRG. Genes expressed in the former but not the latter cDNA populationare specific to trkA⁺ nociceptive neurons.

Total RNA was isolated from the dorsal root ganglia (DRG) of newbornwild type or Ngn1^(−/−) mice (see Ma et al. Genes Develop. 13:1717-1728(1999), Fode et al. Neuron 20:483-494 (1998) and Ma et al. Neuron20:469-482 (1998)). A suppression-PCR-based method (Clontech) was thenused to enrich for genes expressed in wild type but not Ngn1 mutant DRG.Briefly, cDNA was synthesized from the RNA using Superscript reversetranscriptase (Gibco) with oligo dT primers, and was amplified with theSmart PCR Amplification Kit (Clontech). The amplified wild-type andNgn1^(−/−) DRG cDNAs were used as tester and driver, respectively, inthe PCR-Select subtractive hybridization protocol (Clontech).Differential screening by dot blot analysis identified several clones,which were enriched in cDNA from wild-type DRG compared to that fromNgn1^(−/−) DRG. These clones were analyzed further by nucleotidesequencing and in situ hybridization.

Approximately 1,600 positives were identified in the primary screen, andof these 142 were sequenced. Fifty of these represented known genes, and92 represented new genes (see Table 2). Among the known genes wereseveral signaling molecules specifically expressed in nociceptivesensory neurons. These included VR-1, calcitonin gene-related peptide(CGRP), the tetrodotoxin-insensitive sodium channel (SNS-TTXi) anddiacylglycerol kinase. Among the new genes were several encodingproteins with structural features characteristic of ion channels orreceptors, which were revealed by in situ hybridization to bespecifically expressed in a subset of DRG sensory neurons. Thesemolecules are described in more detail in Examples 2 and 3.

TABLE 2 Summary of results of the differential hybridization screeningfor genes involved in pain sensation. # of times isolated from thescreen Name A. Known genes: 13 NaN 9 Diacylglycerol kinase 7Synaptophysin Iia 5 Vanilloid receptor1 3 GluR5-2c 2 CGRP 2 CLIM1 1SNS-TTXi 1 Alpha N-catenin I 1 Brain Na channel III 1 NICA6 1Secretogranin B. Novel genes: 2 Mrg3 (a novel G-protein-coupledreceptor) 2 DRG12 Note: Previous studies have shown that the genes withbolded letters are expressed specifically in nociceptors.

Example 2 A Novel Family of Putative G Protein-Coupled ReceptorsSpecifically Expressed in Nociceptive Sensory Neurons

Among the novel genes isolated from the screen were two independentclones encoding a receptor protein with 7 transmembrane segments (SEQ IDNO: 1), a characteristic of G protein-coupled receptors. The novel 7transmembrane receptor isolated is most closely related to the oncogenemas, and therefore has been named mas-related gene-3 (mrg3). mrg3 isalso known as mas-related gene Al, or MrgA1. A complete coding sequencefor mrg3 has been deduced from the genomic DNA sequence (FIGS. 1B-D andSEQ ID NO: 2). MrgA1 shows significant homology (35% identity) to MAS1(Young et al. Cell 45: 711-9 (1986)). It also shares significanthomology (30-35% identity) with two other mammalian GPCRs, calledMas-related gene 1 (MRG1) (Monnot et al. Mol Endocrinol 5: 1477-87(1991)) and rat thoracic aorta (RTA) (Ross et al. Proc Natl Acad Sci USA87: 3052-6 (1990)).

Such G protein-coupled receptors are expressed in other classes ofsensory neurons, such as olfactory and gustatory neurons, but moleculesin this class had not previously been described in DRG sensory neurons,with the exception of the Protease-Activated Receptors (PARs).

Further screening of mouse DRG cDNA library and mouse genomic library byusing mrg3 DNA as a probe has identified nine additional closely relatedgenes named mrg4 (MrgA2), mrg5 (MrgA3), mrg6, mrg7, mrg8 (MrgA4), mrg9(MrgA5), mrg10 (MrgA6), mrg11 (MrgA7), and mrg12 (MrgA8). Among them,mrg4, 5 and mrg 8-12 contain full-length open reading frames (see FIGS.1A-1D). Two human homologues were found by searching databases using theblast program. The protein alignment of the eight mrg genes, mrg3-8 andhuman1-2, suggested that they define a novel G protein-coupled receptorgene family (FIGS. 1A-1D).

In particular MrgA1-4 were isolated from a P0 mouse DRG cDNA library andclones containing the entire ORFs of MRGsA5-8 were isolated from a mousegenomic BAC library arrayed on filters (Incyte Genomics). FIG. 6A showsan alignment of the polypeptide sequence of MrgA1-8 and indicates thetransmembrane domains as well as the cytoplasmic and extracellularloops. In addition, other mouse MrgAs, as well as other human Mrgsequences, were identified by searching the Celera mouse and human(Venter et al. Science 291: 1304-51 (2001)) genomic databases, using theTBLASTN program with MrgA1 as the query. Table 3 shows that the MrgAgenes are highly homologous to each other. This high degree of homologycombined with the presence of certain characteristic conserved residuesindicates that they define a novel subfamily of the MAS family of GPCRs.

To identify additional members of the mouse Mrg family, TBLASTN searcheswere run against the Celera mouse fragment database (indexed Jan. 7,2001; 18,251,375 fragments) using MRGA1 and MRGA4 protein sequences asqueries. These searches identified 299 unique mouse genomic DNAfragments. The sequences of these fragments were downloaded andassembled into contigs with GELMERGE (GCG Wisconsin Package) understringent conditions (90% identity, 20 nt minimum overlap). GELMERGE wasrun again (80% identity, 20 nt minimum overlap) to reduce the datasetfurther. The consensus nucleotide sequence from each contig was thenqueried against the Celera mouse fragment database with BLASTN toidentify additional sequences for assembly (final n=536 fragments). Theconsensus sequences from the final assembly were placed into a FASTAformatted database. This database was then searched with TFASTY usingMRGA1 as query to identify the potential coding regions from eachconsensus sequence, regardless of whether the error-prone genomicsequence introduced stop codons or frameshifts into the proteins(Pearson, W. R. (1999). Flexible similarity searching with the FASTA3program package. In Bioinformatics Methods and Protocols, S. Misener andS. A. Krawetz, eds. (Totowa, N.J.: Humana Press), pp. 185-219). Theprotein sequences from these searches were then combined into a singleFASTA formatted file for phylogenetic analysis.

Using this analysis, 16 additional members of the murine MrgA subfamilywere identified (FIG. 6B). In addition to this subfamily, two closelyrelated Mrg subfamilies called MrgB and MrgC, were also discovered (FIG.6B). To confirm the existence of an ORF in the mouse MrgB genes,high-fidelity PCR was used to amplify mMrgB1-5, mMrgD, and mMrgE fromC57B1/6 mouse genomic DNA. Several independent clones were sequenced andconfirmed the ORF predictions. The presence of numerous stop codons andframe shifts in the assembled Celera sequence indicated that the mMrgCgenes are pseudogenes.

The MrgB subfamily contains 14 genes, whereas MrgC has 12 members. Thepercent sequence identity within each of these subfamilies is greaterthan 50% (Table 3). Strikingly, all 12 MrgC members appear to bepseudogenes (FIG. 1B, “Ψ”), as they contain multiple premature stopcodons, frameshift mutations or both. Together, therefore, the MrgA andMrgB subfamilies comprise 36 intact ORFs.

TABLE 3 Similarity and identity between murine MRG subfamilies mMRGA1mMRGA2 mMRGA3 mMRGB1 mMRGB2 mMRGB3 mMRGC1 mMRGC2 mMRGC3 mMRGA1 — 77.973.1 48.1 46.3 43.6 44.9 46.7 47.8 mMRGA2 87.5 — 71.8 42.4 45.4 42.741.5 44.5 43.5 mMRGA3 85.1 83.1 — 47.9 46.8 44.2 46.0 49.8 46.6 mMRGB172.1 66.8 70.2 — 57.6 50.0 42.9 47.1 45.3 mMRGB2 68.7 67.7 69.4 72.7 —53.5 41.8 44.4 43.1 mMRGB3 65.2 65.7 64.6 69.5 73.5 — 37.0 38.8 36.4mMRGC1 69.5 65.2 70.9 64.4 67.0 63.3 — 76.0 79.1 mMRGC2 69.8 72.5 74.269.4 70.8 65.7 81.4 — 78.8 mMRGC3 70.9 67.2 71.0 66.2 69.5 64.6 86.186.3 —

Percent identity (top-right, bold) and percent similarity (bottom-left)between the protein sequences are indicated. “hMRG” indicates a humanMRG amino acid sequence; “mMRG” indicates a murine MRG sequence. “hMRGX”is used to indicate a human homolog of mMRGA and mMRGB sequences (FIG.1B). Values were derived from global alignments using the GAP program inthe GCG package.

Searches of the Celera (Venter et al. Science 291: 1304-51 (2001)) andpublic (Consortium. Nature 409: 860-921 (2001)) genomic sequencedatabases, using both BLAST (Altschul et al. Journal of MolecularBiology 215: 403-410 (1990)) and Hidden Markov Models (HMMs (Eddy.Bioinformatics 14, 755-63 (1998)), revealed 4 closely related (˜50%identity) full-length human genes, and at least 10 human pseudogenes.Briefly, TBLASTN searches were run against the Celera human genomedatabase (Venter et al. Science 291: 1304-51 (2001)) using the mMrgA1protein sequence as the query. The genomic sequences that wereidentified in this search were downloaded, placed into a FASTA formatteddatabase and searched with TFASTY to identify a non-redundant set ofproteins. With the exception of hMrgX3, hMrgE, and hMrgΨ8, all humanMrgs were independently identified from a similar analysis of the publichuman genome sequence (Consortium. Nature 409: 860-921 (2001)). HumanMrgX1-4 sequences were independently verified from PCR-amplifiedproducts derived from human BAC clones containing the genes.

Although the human genes appear to be more similar to the murine MrgAsubfamily than the MrgB subfamily in the phylogenetic tree (FIG. 6B,hMrgX1-4), in the absence of clear orthologous pairs we currently referto them as hMrgX genes. In addition to the MrgA, B and C subfamilies, anumber of additional Mas1-related orphan GPCRs were identified by thissearch, including those we refer to as Mrgs D-F (FIG. 6B). Several ofthese sequences, such as MrgD, have clear human orthologs (FIG. 6B,hMrgD). At the protein level hMrgD and mMrgD are 58% identical and 73%similar, while at the nucleotide level they are 73% identical. Alltogether, we identified almost 45 murine and 9 human intact codingsequences belonging to this family.

TABLE 4 Similarity and identity between human and murine MRGs hMRGX2hMRGE mMRGA1 mMRGB4 mMRGB1 mMRGD mMRGE hMRGX2 — 40.2 55.6 50.1 53.4 40.538.8 hMRGE 62.8 — 36.6 32.8 32.8 33.9 76.5 mMRGA1 74.8 57.7 — 48.1 48.137.1 39.7 mMRGB4 71.0 58.0 70.4 — 54.5 34.8 36.6 mMRGB1 73.5 60.5 72.174.1 — 36.5 33.8 mMRGD 61.1 57.6 59.5 64.2 61.3 — 35.1 mMRGE 59.0 84.062.5 63.7 59.1 59.3 —

Percent identity (top-right, bold) and percent similarity (bottom-left)between the protein sequences are indicated. “hMRG” indicates a humanMRG amino acid sequence; “mMRG” indicates a murine MRG sequence. “hMRGX”is used to indicate a human homolog of mMRGA and mMRGB sequences (FIG.1B). Values were derived from global alignments using the GAP program inthe GCG package.

MRG receptors have short (3-21 amino acid) N-termini with no apparentsignal peptide, which are predicted to be located extracellularly. Thetransmembrane domains and intracellular domains are highly conservedsuggesting that the receptors have a shared function. The most divergentregions of MRGA-family receptors appear localized to the extracellularloops (FIG. 6A), suggesting that these receptors recognize differentligands, or the same ligand but with different affinities.Interestingly, we identified 12 single nucleotide polymorphisms in theMrgA1 coding sequence between murine strains C57BL/6J and 129SvJ. These12 changes resulted in 6 amino acid substitutions, all of which wereeither conservative, or which substituted residues expressed at the sameposition by other family members.

A large mouse genomic contig was built by analyzing overlapping BACclones containing MrgA sequences (FIG. 6C). There are 7 MrgA genes,including 3 pseudogenes, residing in this contig. Such clustering is acommon feature of GPCR-encoding gene families (Xie et al. Mamm Genome11: 1070-8 (2000)). Strikingly, all of the human Mrg genes (with theexception of Mas1 and Mrg1) are located on chromosome 11, which alsocontains 50% of all human olfactory receptors genes. All of the MrgAgenes in the murine BAC contig (FIG. 6C) encode intact ORFs withN-terminal methionines, like many other GPCR-encoding genes. Using theCelera mouse genome database, sequences flanking each MrgA coding regionwere obtained and analyzed. This analysis revealed that at least sixMrgA genes have L1 retrotransposon sequences located ˜650 bp downstreamof their coding sequences (FIG. 6B, indicated by “L1”).

All of the eight full-length mas-related genes, mrg3-5 and mrg8-12, areenriched in nociceptive sensory neurons as indicated by their expressionin a subset of DRG sensory neurons which are eliminated in ngn1^(−/−)mutant DRG (FIGS. 2 and 2A).

Example 3 A Novel Two-Transmembrane Segment Protein SpecificallyExpressed in Nociceptive Sensory Neurons

Another novel gene isolated in this screen, drg12 (SEQ ID NO: 13),encodes a protein with two putative transmembrane segments (SEQ ID NO:14). In situ hybridization indicates that, like the mrg genes, this geneis also specifically expressed in a subset of DRG sensory neurons.Although there are no obvious homologies between this protein and othersequences in the database, it is noteworthy that two purinergicreceptors specifically expressed in nociceptive sensory neurons (P₂X₂and P₂X₃) have a similar bipartite transmembrane topology. Therefore itis likely that drg12 also encodes a receptor or ion channel involved innociceptive sensory transduction or its modulation. The hydrophobicityof a homologous region of a drg12 human sequence (SEQ ID NO: 19) iscompared with the hydrophobicity of mouse drg12 in FIG. 4.

Example 4 mrg and drg-12 Genes are Specifically Expressed in NociceptiveSensory Neurons

The prediction of function for mrg-family and drg-12 genes is based ontheir structure and expression pattern, taken together with theidentification of ligands as described below. To determine whether Mrgproteins are expressed in DRG neurons, in situ hybridization usingdioxygenin-labeled riboprobes was performed. Briefly, tissue wasobtained from P0 mouse pups and fixed in 4% paraformaldehyde overnightat 4° C., cryoprotected in 30% sucrose overnight and embedded in OCT.Tissue sections were cut transversely on a cryostat at 18 μm.Non-isotopic in situ hybridization on frozen sections was performed aspreviously described using cRNA probes (Ma et al. Cell 87: 43-52 (1996);Perez et al. Development 126: 1715-1728 (1999)). Eight MrgAs, 5 MrgBsand MrgD were used as probes. At least 10 DRGs were analyzed to countthe number of neurons positive for each probe.

Mrg and drg12 genes, including all eight MrgAs (MrgA1-8), are expressedin subsets of small-diameter sensory neurons in the dorsal root ganglia(DRG) of the mouse (FIG. 7B-I). Importantly, the expression of all eightMrgAs was virtually absent in the DRGs of Ngn1^(−/−) animals (FIG. 7J),consistent with the design of the substractive hybridization screen.Among the eight MrgA clones examined, MrgA1 has the widest expressionwithin sensory neurons in DRGs (13.5%). Other MrgAs are only expressedin several cells per DRG section (ranging from 0.2-1.5% of DRG neurons).This differential abundance may explain why only MrgA1 was isolated inthe original screen. No obvious differences in the expression patternsof MrgA1-8 were noticed in DRGs from different axial levels. Thisexpression is highly specific, in that expression of these genes hasthus far not been detected in any other tissue of the body or in anyother region of the nervous system thus far examined.

Like the MrgA genes, MrgD was also specifically expressed in a subset ofDRG sensory neurons (see below, FIG. 15). In contrast, MrgB1-5 were notdetectably expressed in DRGs. However, mMrgB1 expression has beenobserved in scattered cells in the epidermal layer of skin in newbornmice, as well as in the spleen and the submandibular gland (FIGS. 13 and14). These cells appear to be immune cells that play a role in woundrepair. mMrgB2 also shows this expression pattern. In contrast, mMrgB3,mMrgB4 and mMrgB5 do not appear to be expressed in any of these tissues.

Using Northern blot analysis, human MrgD was found to be expressed inhuman dorsal root gangli neurons. A Northern blot containing 20 μg totalRNA from human DRG neurons was hybridized with a human MrgD probe and atranscript of 4.4 kb was detected. Further analysis indicated that humanMrgD is not expressed in human brain, heart, skeletal muscle, thymus,colon, spleen, kidney, liver, small intestine, placenta, lung orperipheral blood leukocytes. Thus, like mMrgD, human MrgD shows highlyrestricted expression in pain sensing neurons. In addition, the dataindicate that the mouse and human MrgD are functional orthologs.

These results indicate that Mrg and drg12 genes are expressed in primarysensory neurons. However, DRG contain different classes of neuronssubserving different types of sensation: e.g., heat, pain, touch andbody position. Independent identification is provided by the fact thatthe neurons that express the mrg-family and drg12 genes are largely orcompletely eliminated in Ngn1^(−/−) DRG (FIG. 2), because the Ngn1mutation is independently known to largely or completely eliminate thenociceptive (noxious stimuli-sensing) subset of DRG neurons, identifiedby expression of the independent markers trkA, VR-1 and SNS-TTXi (Ma et.al. Genes & Dev. 13: 1717-1728 (1999)). The loss of mrg- anddrg12-expressing neurons in Ngn1^(−/−) mutant DRG therefore indicatesthat these genes are very likely expressed in nociceptive sensoryneurons. Although small numbers of sensory neurons of other classes(trkB⁺ and trkC⁺) are eliminated in the Ngn1^(−/−) mutant as well, mrgand drg12 genes are unlikely to be expressed in these classes of sensoryneurons, because if they were then the majority of mrg- anddrg12-expressing sensory neurons would be predicted to be spared in theNgn1^(−/−) mutant, and that is not the case.

The lack of expression of MrgAs in DRGs from Ngn1^(−/−) mice isconsistent with the idea that they are expressed in cutaneous sensoryneurons. Furthermore, the distribution of MrgA1⁺ cells was similar tothat of neurons expressing trkA, a marker of nociceptive sensory neurons(McMahon et al. Neuron 12: 1161-71 (1994); Snider and Silos-SantiagoPhilos Trans R Soc Lond B Biol Sci 351: 395-403 (1996)) (FIG. 7A, B). Todirectly determine whether MrgA genes are expressed in trkA⁺ cells, insitu hybridization was performed for MrgA1, A3 and A4 in conjunctionwith immunolabeling using anti-trkA antibodies, on neonatal DRG.Fluorescein-UTP-labeled cRNA probes were detected with alkalinephospatase-(AP-) conjugated anti-fluorescein antibody (1:2000, Roche)and developed with Fast Red (Roche) to generate a red fluorescentsignal. After the fluorescent in situ hybridization was performed,sections were incubated in primary antibodies against TrkA (1:5000, giftfrom Dr. Louis Reichardt), VR1 (1:5000, gift from Dr. D. Julius), CGRP(1:500, Chemicon), or SubstanceP (1:1000, Diasorin). All antibodies werediluted in 1×PBS containing 1% normal goat serum and 0.1% TritonX-100.Primary antibody incubations were carried out overnight at 4° C.Secondary antibodies used were goat-anti-rabbit-IgG conjugated to Alexa488 (1:250, Molecular Probes). For double-labeling with Griffoniasimplicifolia IB4 lectin, sections were incubated with 12.5 μg/mlFITC-conjugated IB4 lectin (Sigma) following in situ hybridization.

Double labeling experiment using mrgs antisense RNA probes withanti-trkA antibodies confirmed that mrgs, specifically MrgAs, areco-expressed by trkA+ nociceptive neurons in DRG (see FIG. 7B and FIG.8A-C). Similar results were obtained for MrgD (FIG. 8D). Taken together,these data indicate that MrgAs and MrgD are specifically expressed bynociceptive sensory neurons in DRG.

Further experiments were carried out to determine whether Mrgs areexpressed in particular subsets of nociceptors. Additional doublelabeling experiments using mrgs antisense RNA probes with anit-VR1 andisolectin B4 (IB4)-labeling, as described above, have shown that mrgsare preferentially expressed by IB4+ nociceptive neurons but notVR1-expressing nociceptive neurons (FIGS. 2C and 2D). In particular,combined fluorescent labeling for IB4 together with in situhybridization with MrgA1, A3, A4 and MrgD probes clearly showed thatthese receptors are expressed by IB4⁺ neurons (FIG. 8E-H), and may berestricted to this subset. This result indicates that these Mrgs areexpressed by non-peptidergic nociceptive neurons that project to laminaIIi (Snider and McMahon Neuron 20: 629-32 (1998)). Consistent with thisassignment, the majority (90%) of MrgA1⁺, and all MrgA3⁺, A4⁺ and MrgD⁺cells, lack substance P expression (FIG. 8I-L). Similarly, the majority(70%) of MrgA1⁺, and all MrgA3⁺, A4⁺ and MrgD⁺ cells, do not expressCGRP (FIG. 8M-P), another neuropeptide expressed by C-fiber nociceptors.Previous studies had shown that IB4+ nociceptive neurons were involvedin neuropathic pain resulting from nerve injury (Malmberg, A. B. et al.Science 278: 279-83 (1997)). Neuropathic pain including postherpeticneuralgia, reflex sympathetic dystrophy, and phantom limb pain is themost difficult pain to be managed. Mrgs may play essential roles inmediating neuropathic pain and may provide alternative solutions tomanage neuropathic pain.

Recent studies have provided evidence for the existence of twoneurochemically and functionally distinct subpopulations of IB4⁺nociceptors: those that express the vanilloid receptor VR1 (Caterina etal. Science 288: 306-13 (1997)), and those that do not (Michael andPriestley J Neurosci 19: 1844-54 (1999); Stucky and Lewin J Neurosci 19:6497-505 (1999)). Strikingly, in situ hybridization with MrgA or Dprobes combined with anti-VR1 antibody immunostaining indicated that theMrgA1, A3, A4 and D-expressing cell population was mutually exclusivewith VR1⁺ cells (FIG. 8Q-T). In summary, these expression datademonstrate that MrgA and D genes are expressed in the subclass ofnonpeptidergic cutaneous sensory neurons that are IB4⁺ and VR1⁻ (FIG.9).

MrgA1 is Co-Expressed with Other MrgA Genes

MrgA1 is more broadly expressed than are the other MrgA genes (FIG. 2),suggesting MrgA1 and MrgA2-8 are expressed by different or overlappingsubsets of nociceptors. Double-label in situ hybridization studies usingprobes labeled with digoxigenin and fluorescein indicated that most orall neurons expressing MrgA3 or MrgA4 co-express MrgA1 (FIG. 10A-F).Interestingly, the fluorescent in situ hybridization signals for MrgA3and A4 using tyramide amplification often appeared as dots within nucleithat were circumscribed by the cytoplasmic expression of MrgA1 mRNA,detected by Fast Red (FIG. 10F). Such dots were not observed using theless-sensitive Fast Red detection method, and were only observed in thenuclei of MrgA1⁺ cells. Similar intranuclear dots have previously beenobserved in studies of pheromone receptor gene expression, and have beensuggested to represent sites of transcription (Pantages and Dulac Neuron28: 835-845 (2000)). The results for MrgA1, 3 and 4 indicate that thoseneurons that express the rarer MrgA genes (MrgA2-8) are a subset ofthose that express MrgA1.

To address the question of whether MrgsA2-A8 are expressed in the sameor in different neurons, the number of neurons labeled by single probeswas compared to that labeled by a mixture of all 7 probes (Buck and AxelCell 65: 175-187 (1991)). Approximately 3-fold more neurons (4.5% vs.1%) were labeled by the mixed probe than by an individual probe to MrgA4(FIG. 10J, K), indicating that these genes are not all co-expressed inthe same population of neurons. However, the percentage of neuronslabeled by the mixed probe (4.5%) was less than the sum of thepercentage of neurons labeled by each of the 7 individual probes (6.6%),indicating that there is some overlap in the expression of MrgA2-A8. Inaddition, higher signal intensity was observed in individual neuronsusing the mixed probe, than using a single probe.

Double-labeling experiments with MrgA1 and MrgD probes were alsoperformed. These proteins share only 60% sequence similarity, as shownin FIG. 6B and Table 3. The results of these experiments indicated onlypartial overlap between neurons expressing these two receptors (FIG.10G-I). Approximately 15% (118/786) of neurons expressing either MrgA1or MrgD co-expressed both genes. Thirty-four percent (118/344) of MrgA1⁺cells co-expressed MrgD, while 26.7% (118/442) of MrgD⁺ cellsco-expressed MrgA1.

Taken together, these data indicate the existence of at least threedistinct subpopulations of IB4⁺, VR1⁻ sensory neurons: MrgA1⁺MrgD⁺;MrgA1⁺MrgD⁻ and MrgA1⁻ MrgD⁺. The MrgA1⁺ subset is further subdividedinto different subsets expressing one or more of the MrgsA2-A8.

Mrg-Family Genes Encode Putative G-Protein Coupled Receptors (GPCRs).

Hydrophobicity plots of the encoded amino acid sequences of themrg-family genes predicts membrane proteins with 7 transmembranesegments. Such a structure is characteristic of receptors that signalthrough “G-proteins.” G proteins are a family of cytoplasmic moleculesthat activate or inhibit enzymes involved in the generation ordegradation of “second messenger” molecules, such as cyclic nucleotides(cAMP, cGMP), IP₃ and intracellular free calcium (Ca⁺⁺). Such secondmessenger molecules then activate or inhibit other molecules involved inintercellular signaling, such as ion channels and other receptors.

G protein-coupled receptors (GPCRs) constitute one of the largestsuper-families of membrane receptors, and contain many subfamilies ofreceptors specific for different ligands. These ligands includeneurotransmitters and neuropeptides manufactured by the body (e.g.,noradrenaline, adrenaline, dopamine; and substance P, somatostatin,respectively), as well as sensory molecules present in the externalworld (odorants, tastants).

Although the mrg-family genes are highly homologous, the most divergentregions were the extracellular domains (see FIG. 6A). The variability ofthe extracellular domains of mrg family suggests that they may recognizedifferent ligands.

The fact that the mrg-family genes encode GPCRs, and are specificallyexpressed in nociceptive sensory neurons, suggest that these receptorsare involved, directly or indirectly, in the sensation or modulation ofpain, heat or other noxious stimuli. Therefore the mrg-encoded receptorsare useful as targets for identifying drugs that effect the sensation ormodulation of pain, heat or other noxious stimuli. The nature of themost useful type of drug (agonistic or antagonistic) will reflect thenature of the normal influence of these receptors on the sensation ofsuch noxious stimuli. For example, if mrg-encoded receptors normally actnegatively, to inhibit or suppress pain, then agonistic drugs wouldprovide useful therapeutics; conversely, if the receptors normally actpositively, to promote or enhance pain, then antagonistic drugs wouldprovide useful therapeutics. There might even be certain clinicalsettings in which it would be useful to enhance sensitivity to noxiousstimuli, for example in peripheral sensory neuropathies associated withdiabetes.

The nature of the influence of mrg-encoded GPCRs on pain sensation maybe revealed by the phenotypic consequences of targeted mutation of thesegenes in mice. For example, if such mice displayed enhanced sensitivityto noxious stimuli, then it could be concluded that the receptorsnormally function to inhibit or suppress pain responses, and vice-versa.Alternatively, high-throughput screens may be used to identify smallmolecules that bind tightly to the mrg-encoded receptors. Such moleculeswould be expected to fall into two categories: agonists and antagonists.Agonists would be identified by their ability to activate intracellularsecond messenger pathways in a receptor-dependent manner, whileantagonists would inhibit them. Testing of such drugs in animal modelsof pain sensitivity will then reveal further information concerning thefunction of the GPCRs: for example, if the molecules behave as receptorantagonists in vitro, and they suppress sensitivity or responsiveness tonoxious stimuli in vivo, then it may be concluded that the receptornormally functions to promote or enhance pain sensation. Conversely, ifreceptor agonists suppress, while antagonists enhance, pain sensation invivo, then it may be concluded that the receptor normally functions tosuppress or inhibit pain sensation.

drg12 Encodes a Putative Transmembrane Signaling Molecule

Hydrophobicity plots of the encoded amino acid sequence of the drg12gene predicts a membrane protein with 2 transmembrane segments. Themembrane localization of this protein has been verified byimmuno-staining of cultured cells transfected with an epitope-taggedversion of the polypeptide. Although the DRG12 amino acid sequence hasno homology to known families of proteins, its bipartite transmembranestructure strongly suggests that it is involved in some aspect ofintercellular signaling, for example as a receptor, ion channel ormodulator of another receptor or ion channel. This prediction issupported by the precedent that two known receptors with a similarbipartite transmembrane topology, the purinergic P₂X₂ and P₂X₃receptors, are like DRG12, specifically expressed in nociceptive sensoryneurons.

Based on this structural data, and its specific expression innociceptive sensory neurons, it is probable that DRG12 is involved,directly or indirectly, in the sensation or modulation of noxiousstimuli. Accordingly, the drg12-encoded protein is a useful target forthe development of novel therapeutics for the treatment of pain.

Example 5 Mrg Proteins are Receptors for Neuropeptides

As discussed above, the structure of the proteins encoded by Mrg genesindicates that they function as receptors. To identify ligands for theMrg receptors, selected MrgA genes were tested in a calcium releaseassay. MrgA genes, including MrgA1 and MrgA4, were cloned into aeukaryotic expression vector and transfected into human embryonic kidney(HEK) 293 cells. HEK-293 cells were obtained from the ATCC and culturedin DMEM supplemented with 10% fetal bovine serum. An HEK293-Gα₁₅ cellline stably expressing Gα₁₅ was provided by Aurora BiosciencesCorporation and grown on Matrigel™ (growth factor reduced Matrigel,Becton Dickinson, diluted 1:200 with serum-free DMEM)-coated flasks andmaintained at 37° C. in DMEM (GibcoBRL) supplemented with 10%heat-inactivated fetal bovine serum, 2 mM L-glutamine, 0.1 mMnon-essential amino acids, 1 mM sodium pyruvate, 25 mM HEPES and 3 μg/mlblastcidin-S. For transfection, cells were seeded on Matrigel-coated 35mm glass-bottom dishes (Bioptech Inc., Butler, Pa.). After 16-24 hr,cells were transfected using FuGENE 6 (Roche). Transfection efficiencieswere estimated by visualization of GFP fused to the C-terminus of MrgA1and A4, and were typically >60%. Fusing GFP to the C-termini of the MrgAcoding sequences additionally allowed for visual confirmation of theintracellular distribution of the receptors and their membraneintegration in the transfected cells (FIG. 11D).

To increase the sensitivity of the calcium release assay, in someexperiments the MRGA-GFP fusion proteins were expressed in HEK 293 cellsmodified to express Gα₁₅, which couples GPCRs to a signal transductionpathway leading to the release of intracellular free Ca²⁺ (Offermann andSimon J Biol Chem 270: 15175-80 (1995)). This calcium release can bemonitored ratiometrically using Fura-2 as a fluorescent indicator dye(Tsien et al. Cell Calcium 6: 145-57 (1985)) (FIG. 11A-C). Thisheterologous expression system has been previously used to identifyligands for taste receptors (Chandrashekar et al. Cell 100: 703-11(2000)).

Because MRGAs exhibit the highest sequence similarity to peptide hormonereceptors, approximately 45 candidate peptides were screened for theirability to activate MRGA1, using the intracellular Ca²⁺-release assay.Briefly, transfected cells were washed once in Hank's balanced saltsolution with 11 mM D-glucose and 10 mM HEPES, pH 7.4 (assay buffer) andloaded with 2 μM Fura-2 AM (Molecular Probes) at room temperature for 90min, with rotation. Loaded cells were washed twice with assay buffer andplaced on a micro-perfusion chamber (Bioptech). The chamber was mountedon top of a Olympus IMT2 inverted microscope, and imaged with an OlympusDPlanApo 40× oil immersion objective lens. Samples were illuminated by a75W xenon bulb, and a computer-controlled filter changer (Lambda-10;Shutter Instruments) was used to switch the excitation wavelength. Acooled CCD camera (Photometric) was used in detecting fluorescence.GFP-positive cells within a field were identified using an excitationwavelength of 400 nm, a dichroic 505 nm long-pass filter and an emitterbandpass of 535 nm (Chroma Technology). In the same field, calciummeasurements were performed at an excitation wavelength of 340 nm and380 nm, and an emission wavelength of 510 nm. Agonists were diluted inassay buffer and solution changes accomplished by micro-perfusion pump(Bioptech). Fura-2 fluorescence signals (340 nm, 380 nm and the 340/380ratio) originating from GFP-positive cells were continuously monitoredat 0.4- or 1-second intervals and collected using Axon Imaging Workbench4.0 software (Axon). Instrument calibration was carried out withstandard calcium solutions (Molecular probes) in glass bottom dishes(MatTek Corp.).

At a concentration of 1 μM, numerous neuropeptides produced some levelof activation of MrgA1-expressing cells (FIG. 12A). These included ACTH,CGRP-I and -II, NPY and somatostatin (SST). Nevertheless, many otherpeptide hormones did not activate MRGA1, including angiotensins I-IIIand neurokinins A and B, alpha-MSH and gamma2-MSH (FIG. 12A and data notshown). MrgA1 was only very weakly activated by ecosanoid ligands suchas Prostaglandin-E1 and Arachidonic Acid (data not shown).

The most efficient responses in MrgA1-expressing HEK cells were elicitedby RFamide peptides, including FLRF and the molluscan cardioactiveneuropeptide FMRFamide (Price and Greenberg Science 197: 670-671 (1977))(Phe-Met-Arg-Phe-amide) (FIG. 11C, 12A). Two mammalian RFamide peptides,NPAF and NPFF, which are cleaved from a common pro-peptide precursor(Vilim et al. Mol Pharmacol 55: 804-11 (1999)) were then tested. Theresponse of MrgA1-expressing cells to NPFF at 1 μM was similar to thatseen with FMRFamide, while that to NPAF was significantly lower (FIG.12A). MrgA1 was also weakly activated by two other RFamide ligands,γ₁-MSH and schistoFLRF (data not shown).

In order to examine further the specificity of activation of MrgA1 andA4, the top candidate ligands emerging from the intial screen weretested on these same receptors expressed in HEK cells lacking Gα₁₅.MrgA1 and A4 expressed in this system retained responses to RFamidepeptides (FIG. 12B, C), demonstrating that the intracellular Ca²⁺release responses seen in the initial screen are not dependent on thepresence of exogenous Gα₁₅. This indicates that MrgAs act in HEK cellsvia Gq or Gi. The response of MrgA1-expressing HEK cells to NPFF waslower than that to FLRF (FIG. 12B), and there was no response to NPAF.Conversely, MrgA4-expressing cells responded to NPAF, but not to NPFF orFLRF (FIG. 12C). In both cases, the response to NPY seen inGα₁₅-expressing cells (FIG. 11A) was lost completely, while those toCGRP-II and ACTH were considerably diminished.

In order to determine the lowest concentrations of RFamide ligandscapable of activating MrgA1 and A4, dose-response experiments werecarried out in HEK cells expressing Gα₁₅, which afforded greatersensitivity (FIG. 12D, E). These experiments indicated that MrgA1 couldbe activated by FLRF at nanomolar concentrations (FIG. 12D; EC₅₀≈20 nM),and by NPFF at about an order of magnitude higher concentration (FIG.12D; EC₅₀≈200 nM), whereas NPAF was much less effective. In contrast,MrgA4 was well activated by NPAF (FIG. 12E; EC₅₀≈60 nM), and much moreweakly activated by FLRF and NPFF. Neither receptor showed strongactivation in response to RFRP-1, -2 or -3, a series of RFamide ligandsproduced from a different precursor (Hinuma et al. Nat Cell Biol 2:703-8 (2000)). These data confirm that MrgA1 and MrgA4 display differentselectivities towards different RFamide ligands in this system. Bycontrast, these receptors responded similarly to ACTH (EC₅₀˜60- and 200nM for MrgA1 and A4, respectively; data not shown).

Finally, given the sequence similarity between MRGA receptors and MAS1,the responsiveness of cells expressing exogenous Mas1 to NPFF, NPAF andFLRF was tested. MAS1 showed a profile distinct from both MrgA1 andMrgA4 (FIG. 12F): like MrgA1, it was activated by NPFF at a similarconcentration of the peptide (EC₅₀≈400 nM), but unlike MrgA1 it waspoorly activated by FLRF. In contrast to MrgA4, MAS1 did not respondwell to NPAF. No response was detected in MAS1-expressing cells uponexposure to Angiotensins I and II, ligands which have been previouslyreported to activate this receptor (Jackson, T. R., et al. Nature 335:437-40 (1988)). Nor did MAS1 respond to ACTH. Thus, MAS1, MrgA1 andMrgA4 expressed in this heterologous system are all activated by RFamidefamily ligands, but with differing ligand-sensitivities and-selectivities (Table 4).

TABLE 4 Selectivity of activation of Mas-related GPCRs by RF-amideligands in HEK cells A. Ligand receptor FLRF NPFF NPAF MRGA1 +++ ++ +/−MRGA4 +/− +/− +++ MAS1 +/− ++ +/−

Relative efficacy of activation of the indicated receptors by theindicated ligands is shown. For quantification, see FIG. 6. “+++”indicates 10 nM<EC₅₀<100 nM; “++” indicates 100 nM<EC₅₀<500 nM; “+/−”indicates weak response seen at 1 μM. For details see FIG. 6.

A novel family consisting of close to 50 MAS1 related g-protein coupledreceptors has been identified. The specific expression of severalclasses of these receptors in a subset of nociceptive sensory neuronsindicates that these receptors play a role in the sensation ormodulation of pain. Consistently, these receptors have been shown to beactivated by RFamide neuropeptides, which are known to mediateanalgesia. As a result, these receptors provide a novel target foranti-nociceptive drugs.

Although the present invention has been described in detail withreference to examples above, it is understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the following claims. Allcited patents, patent applications and publications referred to in thisapplication are herein incorporated by reference in their entirety.

What is claimed is:
 1. A method for identifying a compound useful in altering sensory perception in a mammal comprising the steps of: a) expressing a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 in a host cell capable of producing a second messenger response; b) contacting the host cell with one or more test compounds; c) measuring the second messenger response in the host cell; and d) identifying compounds that increase the measured second messenger response as compounds that are useful in altering sensory perception in a mammal.
 2. The method of claim 1 wherein said host cell is a eukaryotic cell.
 3. The method of claim 2 wherein the eukaryotic cell is a hamster embryonic kidney (HEK) cell.
 4. The method of claim 3 wherein said HEK cell expresses Gα₁₅.
 5. The method of claim 1 wherein measuring a second messenger response comprises measuring a change in intercellular calcium concentration.
 6. The method of claim 5 wherein said change in intercellular calcium concentration is measured with FURA-2 calcium indicator dye.
 7. The method of claim 1 wherein measuring a second messenger response comprises measuring the flow of current across the membrane of the cell.
 8. The method of claim 1 wherein said sensory perception is the perception of pain.
 9. A method for identifying a compound useful in treating impaired sensory perception in a mammal comprising the steps of: a) expressing a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 in a host cell capable of producing a second messenger response; b) contacting the host cell with an RFamide peptide; c) contacting the host cell with one or more test compounds; d) measuring the second messenger response in the host cell; and e) identifying compounds that alter the measured second messenger response to the RFamide peptide as compounds that are useful in treating impaired sensory perception in a mammal.
 10. The method of claim 9 wherein the impaired sensory perception is pain. 